263

KeywordsCentral Italy,Monitoring,Mosquitoes.

SummaryThis study reports the mosquito collections conducted in July‑August 2013 in Castiglione del Lago (Umbria Region, Italy) during the entomological surveillance within the West Nile national control program. The mosquito collections showed the noteworthy occurrence of Anopheles hyrcanus s.l. (n = 156; 35.8% of the whole sample), a relatively rare mosquito species in Italy, with molecular analyses confirming that these mosquitoes belong to An. hyrcanus s.s. The same catching site was characterized by a relevant richness in mosquito species, in particular the following were found: Anopheles maculipennis s.l. (n = 146; 33.5% composed by 80% Anopheles melanoon, 13.3% Anopheles maculipennis s.s. and 6.7% Anopheles atroparvus), Culex pipiens s.l. (n = 116; 26.6% composed by 90% Cx. p. pipiens, 5% Cx. p. molestus and 5% Cx. p. pipiens/molestus), Culiseta annulata (n = 10; 2.3%), Aedes albopictus (n = 3; 0.7%), and Anopheles claviger (n = 5; 100%).

Veterinaria Italiana 2016, 53 (3), 263‑266. doi: 10.12834/VetIt.668.3283.3Accepted: 17.11.2015 | Available on line: 03.08.2016

1 Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy.2 Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Campo Boario, 64100 Teramo, Italy.

3 Unità Sanitaria Locale Umbria ‑ 1, Via Carducci 15, 06127 Castiglione del Lago (PG), Italy.* Corresponding author at: Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy.

Tel.: +39 06 4990 2301, e‑mail: luciano.toma@iss.it.

Luciano Toma1*, Monica Catalani2, Antonella Catalano3,Maria Goffredo2, Roberto Romi1 and Marco Di Luca1

Finding of Anopheles (Anopheles) hyrcanus(Pallas, 1771) (Diptera, Culicidae)

during the entomological surveillancefor West Nile virus in Umbria, Italy

Parole chiaveItalia centrale,Monitoraggio,Zanzare.

RiassuntoIl presente studio riporta i risultati delle catture di zanzare effettuate tra giugno e agosto 2013 in Umbria nel Comune di Castiglione del Lago (PG), nell’ambito delle attività entomologiche del Piano Nazionale di sorveglianza per la West Nile. Tra le catture effettuate con una trappola CDC e con una trappola BG‑Sentinel®, ha destato particolare interesse il rinvenimento di Anopheles hyrcanus s.l. (n  =  156; 35,8%), specie poco comune in Italia. L’analisi molecolare e lo studio delle sequenze hanno confermato l’appartenenza delle zanzare esaminate alla specie An. hyrcanus s.s. Una certa ricchezza di fauna culicidica è stata riscontrata nello stesso sito di cattura, per il rinvenimento di Anopheles maculipennis s.l. (n = 146; 33,5% di cui 80% Anopheles melanoon, 13,3% Anopheles maculipennis s.s. and 6,7% Anopheles atroparvus), Culex pipiens s.l. (n = 116; 26,6% di cui 90% Cx. p. pipiens, 5% Cx. p. molestus e 5% Cx. p. pipiens/molestus), Culiseta annulata (n = 10; 2,3%), Aedes albopictus (n = 3; 0,7%) e Anopheles claviger (n = 5; 100%).

Rinvenimento di Anopheles (Anopheles) hyrcanus (Pallas, 1771)(Diptera, Culicidae) durante la sorveglianaza entomologica

per il virus West Nile virus in Umbria

SHORT COMMUNICATION

264 Veterinaria Italiana 2017, 53 (3), 263‑266. doi: 10.12834/VetIt.668.3283.3

Anopheles hyrcanus reported in Umbria, Italy Toma et al.

breeding site of this mosquito, probably contributed to the decrement of this species, which is actually uncommon in Italy (Romi et al. 1997).

Within the Anopheles Hyrcanus Group, some species are considered relevant for human health as vectors of malaria and other mosquito‑borne diseases in the Oriental and Palearctic regions. Some authors assume An.  hyrcanus  s.s. to be involved in malaria transmission particularly in the Northern Afghanistan (Ramsdale 2001, Ponçon et  al. 2007). In the countries of Central Asia, it is considered a secondary malaria vector. Anopheles hyrcanus s.s. can easily be infected with Plasmodium vivax; whereas it is less commonly infected with P. malariae, and even less with P. falciparum (WHO 1990). In addition, the vectorial capacity for Sindbis and Thayna viruses has been demonstrated in this species and its role of vector for dirofilariases is suspected (Hubálek 2008, Azari‑Hamidian et al. 2009). According to some other authors, the high level of anthropophily observed in An.  hyrcanus contributes to the importance of this insect as vector of pathogens for humans (Ramsdale 2001, Ponçon et al. 2007).

Mosquito females were collected from June to August 2013 by 1 CDC Miniature Light/CO2 baited Trap (2 days catch monthly) and 1 BG‑Sentinel® BG‑Lure® baited Trap (1 day catch in July). A total of 436  mosquitoes from 6 catches were morphologically identified (Severini et  al. 2009), as belonging to the following species: Anopheles hyrcanus s.l., An.  maculipennis  s.l., An.  claviger/petragnani, Culex pipiens s.l., Culiseta annulata (Schrank, 1776) and Aedes albopictus (Skuse, 1897) (Figure 1). Where morphological analysis led to identify species complexes, molecular investigations allowed for completing the identification at species level.

Molecular analysis were carried out on specimens belonging to An. maculipennis s.l. using the rRNA

The surveillance National Plan for West Nile Virus in Italy is ongoing since 2002, and it includes mosquito collections carried out in specific study areas. The present study reports the results of mosquito catches made from June to August 2013 in Umbria region, Castiglione del Lago Municipality, Perugia province, Central Italy. The study site consists of a farm with horses and cattle in locality La Pievaccia (43°06’11.32’’N; 11°55’47.75’’E), near Montepulciano Lake. Given the naturalistic and faunistic richness, this area is identified as National Important Bird Area (IBAs). Catches from this site were characterized by an unusual density of the species Anopheles (Anopheles) hyrcanus s.l. (Pallas, 1771). Anopheles hyrcanus, belonging to the Hyrcanus Group, which includes 26 recognised species, it has an Eastern Palaearctic distribution, extending from the Iberian Peninsula, across southern Europe (South of the Alps) and Asia (South of about 50°N), to the Pacific (Ramsdale and Snow 2000).

Widely studied in East European and Middle East Countries, but poorly investigated elsewhere (Ramsdale 2001), An. hyrcanus was first considered as a largely distributed polymorphic species with differences in adult morphology and behaviour (Gutsevich et  al. 1971, Gutsevich 1976). After an intricate review process, Ramsdale (Ramsdale 2001) listed 3 species for the Western Palaearctic Region: An.  hyrcanus, Anopheles pseudopictus Grassi, 1899, elevated to species status by Glick (Glick 1992), and Anopheles chodukini Martini, 1929, which was described from a unique female specimen in Uzbekistan in 1929. Recently, genetic analyses suggested that An.  hyrcanus and An. pseudopictus belong to the same species (Ponçon et  al. 2008). The authors revealed no genetic differences between the 2 sympatric taxa from Camargue (France) using different DNA markers (ITS‑2 and D3 of 28S ribosomal DNA and COI and COII genes of mitochondrial DNA). In the same study, a genetic distinction between French and Turkish An.  hyrcanus was shown only by COI and COII comparison, and it was ascribed to the geographical distance between the 2 populations.

The Western distribution of An.  hyrcanus includes several Southern European countries (Ramsdale and Snow 2000). However, in the last decade it has reached also Slovakia (Halgoš and Benkova 2004), new records of An.  hyrcanus in Southern Moravia indicate the Northern point of its occurrence in Europe (Halgoš and Benkova 2004, Votypka et  al. 2008). Common in Italy until some decades ago, the presence of this species dramatically shrank in the last years probably because of the gradual disappearing of original larval breeding sites once very common in wet areas as ponds, marshes, puddles, and flooded fields (Romi et al. 1997). Moreover, control activities carried out in rice‑fields, the main artificial larval

Anopheles hyrcanus s.l.

36%

Anopheles maculipennis s.l.

33% Culex pipiens 27%

Culisetaannulata

2%

Anopheles claviger/petragnani

1%Aedesalbopictus

1%

Figure 1. Morphological identification of mosquito species collected in Umbria Region in June‑August 2013: the percentage of the single species out of the 454 specimens is here reported.

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Toma et al. Anopheles hyrcanus reported in Umbria, Italy

occurrence from the Culex pipiens sample, while the CQ11 marker identified 20 specimens as Cx. pipiens f. pipiens Linné, 1758 (n = 18), Cx. pipiens f. molestus Forskal, 1775 (n = 1), and their hybrid (n = 1).

The most interesting aspect of this study was the finding of An. hyrcanus s.s. species collected in July and August 2003 only, indicating the presence of an abundant local population very close to the collection site.

According to dedicated literature, the molecular analysis confirms An.  hyrcanus  s.s. as the only species of the Anopheles Hyrcanus Group occurring in Italy, dismissing the hypothesis of an accidental introduction or spread of other components of the complex from the Eastern regions of its distribution range. Some specimens of An.  hyrcanus  s.l. were found for the first time in Austria, during an entomological survey in Donau‑Auen National Park in the surrounding of Vienna (Lebl et al. 2013). In this case, it was impossible to draw conclusions about the taxonomy of this finding, as no molecular investigation was conducted. We consider the data reported in this study as very interesting under the faunistic aspect, not only for the occurrence of An. hyrcanus s.s., but also for the other species found in association (An. maculipennis s.s., An. claviger, Cs. longiareolata and Ae. albopictus), which are very common but typical for peridomestic and rural environments. The occurrence of An. atroparvus known as the Northern vicariant species of An. labranchiae Falleroni, 1926, formerly considered one of the main malaria vectors, confirms what already reported by previous entomological studies in Central Italy (Toma et al. 2008, Di Luca et al. 2009).

In such contest, the occurrence of An. melanoon and An. maculipennis s.s., mainly zoophilic species, seems to be linked to the presence of horses in the collection site. Concerning Cx. pipiens, the main West Nile virus vector in Europe (Rizzoli et  al. 2015  a,  b, Rudolf et  al. 2014), the finding of only 1 specimen of the molestus form and 1 hybrid together to the rural pipiens form, points out the occurrence of an autogenic and anthropophilic fraction within Cx. pipiens population.

The diversity of mosquito species observed in this site, prompts us to conduct further studies on its faunistic composition, and to focus in particular on those species which are of zoonotic relevance.

This study was partially founded by EU grant HEALTH.2010.2.3.3‑3 project 261391 EuroWestNile.

internal transcribed spacer 2 (ITS‑2) as marker, according to Proft and colleagues (Proft et  al. 1999). We used both ACE and CQ11 assays (Smith and Fonseca 2004, Bahnck and Fonseca 2006) to discriminate the species (Cx. torrentium/Cx. pipiens) and the biological forms (Cx. pipiens f. pipiens and Cx.  pipiens f. molestus) within the Cx. pipiens complex. The sequencing analysis of the ITS‑2 was performed for An.  hyrcanus and An. claviger complexes (Severini et al. 2004) (Figure 2).

A multiplex polymerase chain reaction (PCR) for ITS‑2 was conducted on 15 An.  maculipennis  s.l. specimens and permitted to identify 12 specimens of An. melanoon Hackett, 1934, 2 An. maculipennis s.s. Meigen, 1818 and 1 An. atroparvus Van Thiel, 1927. The ITS‑2 sequencing of 20 An. hyrcanus s.l. specimens (GenBank accession No. KP749440‑KP749459) and the following comparison with the sequences available for the Hyrcanus Group in the Western Palaearctic Region (An.  hyrcanus from Uzbekistan AY515173‑AY515177; An.  hyrcanus from France AM773808; An. hyrcanus from Turkey AM773809 and An. pseudopictus from France AM773810) identified the Italian mosquitoes as belonging to the nominal species of the group, An.  hyrcanus  s.s. By the same approach, we identified all the 5 specimens of An. claviger/petragnani as An. claviger (Meigen, 1804) (GenBank accession No. KP749460‑KP749464). The ACE assay excluded the Culex torrentium

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Figure 2. Molecular identification of 15 specimens of An. maculipennis s.l., 20 An. hyrcanus s.l., 20 Cx. pipiens and 5 An. claviger/petragnani collected in Umbria Region in June‑August 2013. Histograms show the number of the specimens (y axis) and their species identification within the complexes of species (x axis).

266 Veterinaria Italiana 2017, 53 (3), 263‑266. doi: 10.12834/VetIt.668.3283.3

Anopheles hyrcanus reported in Umbria, Italy Toma et al.

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