First Nationwide Surveillance of Culex pipiens Complex and Culex torrentium Mosquitoes Demonstrated the Presence of Culex pipiens Biotype pipiens/molestus Hybrids in Germany

Abstract

Mosquitoes and other arthropods may transmit medically important pathogens, in particular viruses such as West Nile virus. The presence of suitable hosts and competent vectors for those zoonotic viruses is essential for an enzootic transmission, which is a prerequisite for epidemics. To establish reliable risk projections, it is an urgent need for an exact identification of mosquito species, which is especially challenging in the case of sibling species, such as Culex. pipiens pipiens biotypes pipiens and molestus. To facilitate detection of different Culex pipiens forms and their hybrids we established a multiplex real-time PCR. Culex pipiens samples were obtained by egg raft collection and rearing until imago stage or adult sampling using CO2 baited traps and gravid traps. In total, we tested more than 16,500 samples collected all over Germany in the years 2011 and 2012. The predominant species in Germany are Culex pipiens pipiens biotype pipiens and Culex. torrentium, but we also detected Culex pipiens pipiens biotype molestus and hybrids of the two pipiens biotypes at sites where both species occur sympatrically. This report of a potentially important bridge vector for West Nile virus might have major impact in the risk projections for West Nile virus in Germany.

The role of Culex pipiens mosquitoes in transmission of West Nile virus in Europe

“…pipiens, during summer (Hesson et al 2013, Rudolf et al 2013, Weitzel et al 2011, WNV outbreaks have not yet occurred in northern Europe. The question, therefore, remains which factors are limiting the transmission of WNV in northern Europe.…”

“…The real-time PCR assay as described by Rudolf et al (2013) was adjusted to optimize the design, because amplification curves did not all show an exponential increase and did not reach the same plateau, which made the interpretation of results difficult. DNA sequences of microsatellite CQ11 of Cx.…”

“…pipiens forward and reverse primers, respectively. For identification of biotype molestus, probe Cpp_mol_P (5'-FAM-TGAACCCTCCAGTAAGGTA-MGB-3') was designed, which is a shorter version of the original probe by Rudolf et al (2013), conjugated with a minor groove binder (MGB) group. The two probes Cpp_pip_ P1 (5'-VIC-CACACAAAYCTTCACCGAA-MGB-3') and Cpp_pip_P2 (5'-VIC-ACACAAACCTTCATCGAA-MGB-3') were designed for identification of biotype pipiens to cover the sequence variation at the original probe binding site.…”

“…torrentium identification. Based on the original primers and probes of Rudolf et al (2013), forward and reverse primers were enlarged at the 5' end to obtain Cx_tor_F (5'-CTTATTAGTATGACACAGGACGACAGAAA-3') and Cx_tor_R (5'-GCATAAACGCCTACGCAACTACTAA-3'). The probe was shortened at the 5' end and conjugated with a MGB group to get Cx_tor_P (5'-FAM-ATGATGCCTGTGCTACCA-MGB-3').…”

“…The view of spatially separated populations of the two biotypes , therefore, may not reflect the real situation in northern Europe, because differentiation between biotypes was not addressed in many studies. Rudolf et al (2013) performed analyses to the biotype level and found that the molestus and pipiens biotypes occur sympatrically in Germany. No strong conclusions on the occurrence of biotype molestus or hybrid frequencies could be drawn, however, because samples were analysed in pools and habitats were not systematically analysed (Rudolf et al 2013).…”