Núñez et al. Parasites Vectors (2019) 12:363 https://doi.org/10.1186/s13071-019-3620-7 Parasites & Vectors SHORT REPORT Open Access European Aedes caspius mosquitoes are experimentally unable to transmit Zika virus Ana I. Núñez1, Sandra Talavera1, Carles Aranda1,2, Lotty Birnberg1, Raquel Rivas1, Núria Pujol1, Marta Verdún1, Anna‑Bella Failloux3 and Núria Busquets1* Abstract Background: Aedes caspius (Pallas, 1771) is a floodwater mosquito species widely distributed in the Western Palae‑ arctic. As an anthropophilic species, its role as an arbovirus vector may be the key for understanding the transmission cycle of certain diseases in Europe such as Zika virus (ZIKV). Concerning vector competence for ZIKV, studies related to Ae. caspius are still scarce. ZIKV is an arbovirus that has provoked a widespread epidemic in the Pacific region (2007– 2013) and in the Americas (2015–2016). ZIKV is associated with serious neurological injuries (e.g. microcephaly) and Guillain-Barré syndrome. Due to the ZIKV epidemics in the American continent, some viraemic travellers coming from endemic countries have been reported in Europe. More knowledge is therefore required to define the susceptibility of autochthonous mosquito species such as Ae. caspius for ZIKV in order to improve arbovirus surveillance and control programmes. In the present study, the vector competence of a European population of Ae. caspius was evaluated for two ZIKV lineages, the Suriname ZIKV strain (Asian lineage) and the MR766 ZIKV strain (African I lineage). Females were tested at 7, 14 and 21 days post-exposure (dpe) to infectious blood meals. An Ae. aegypti PAEA strain was used as a positive control. Results: Aedes caspius presented low susceptibility to ZIKV infection and the virus was only detected by RT-qPCR in body samples. Low viral loads were detected for the MR766 strain at 7 dpe and for the Suriname strain at 14 and 21 dpe. Aedes caspius was unable to produce a disseminated infection and virus transmission at any of the tested time points. Using Ae. aegypti PAEA strain, infection, dissemination and transmission rates were calculated for the Suriname ZIKV strain (Asian lineage) at each time point. For the MR766 ZIKV strain (African I lineage), while only infection rates were estimated at each time point, no dissemination or transmission were detected in either species. Conclusions: The results of the present study reveal that the tested Ae. caspius population has a strong midgut escape barrier that limits the dissemination or transmission of the virus. As such, it seems unlikely that European Ae. caspius mosquitoes could be involved in ZIKV transmission if ZIKV was introduced into Europe. This information may help in designing a better strategy to European surveillance and control programmes for ZIKV. Keywords: Aedes caspius, Saliva, Transmission, Arbovirus, Vector competence, Zika virus *Correspondence: nuria.busquets@irta.cat 1 Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), 08193 Barcelona, Spain Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Núñez et al. Parasites Vectors (2019) 12:363 Page 2 of 7 Background improve arbovirus surveillance and control programmes. Aedes caspius (Pallas, 1771) is a floodwater mosquito For this reason, in the present study we evaluated the species widely distributed in the Western Palaearctic vector competence of an Ae. caspius mosquito popula- [1]. This mosquito species is tolerant to varying levels tion from El Prat de Llobregat (Catalonia, Spain) for two of salinity in larval breeding places [2] and so is present ZIKV lineages, Suriname (Asian lineage) and MR766 in different habitats, including coastlands, irrigation (African I lineage) to measure its potential role in ZIKV channels, swamps and rice fields. Aedes caspius is an transmission. anthropophilic species and a crepuscular feeder and is known to bite during the day and night [3]. These mos- Methods quitoes usually feed aggressively on humans and animals, Mosquito rearing both indoors and outdoors [4]. Thus its role as an arbovi- Aedes caspius larvae were collected from El Prat de Llo- rus vector may be the key for the knowledge of transmis- bregat (Catalonia, Spain) in October 2017. Larvae were sion cycle of certain diseases in Europe, especially given reared in trays containing dechlorinated water supple- its anthropophilic behavior. The vector competence of mented with fish food (Goldfish, Tetra GmbH, Melle, some Ae. caspius populations exposed to different arbo- Germany) until the adult stage. Emerging adults were viruses has been tested in previous studies. Aedes caspius maintained at 26/22 °C (day/night) to simulate summer populations from the Camargue (France) were experi- environmental conditions at the latitude where the mos- mentally found to be potential vectors of arboviruses quitoes were captured, a relative humidity of 80% and a such as chikungunya virus (CHIKV) [5] and Rift Val- light/dark photocycle of 14:10 h. Mosquitoes were fed ad ley fever phlebovirus (RVFV) [6]. However, Ae. caspius libitum with 10% sucrose solution. The F0 generation was from Camargue and from Andalusia (Spain) were unable used for experimental infection. An Ae. aegypti PAEA to transmit West Nile virus (WNV) [7] and Zika virus strain, from Paea (Tahiti, French Polynesia), colonized (ZIKV) [8], respectively. since 1994, was reared in the same conditions and used The ZIKV is an arthropod-borne virus belonging to the as a positive control of ZIKV vector. genus Flavivirus (family Flaviviridae). The virus is pri- marily transmitted in a zoonotic cycle between mosqui- Virus production and titration toes and non-human primates in Africa, although sexual Suriname and African MR766 ZIKV strains provided [9] and perinatal [10] ZIKV transmission have also been by the European EVAg project were used in the present confirmed in humans. The virus has been associated with work. The Suriname ZIKV strain (EVAg no. 011V-01621; severe clinical manifestations and congenital malforma- Asian lineage) was isolated from a placental material of a tions including microcephaly [11] and Guillain-Barré patient in Netherlands in 2016 who came from Suriname syndrome [12]. ZIKV was isolated for the first time from during the ZIKV epidemic [29] and the African MR766 a rhesus macaque monkey in the Zika forest (Uganda) in ZIKV strain (African I lineage) was isolated from a rhe- 1947 [13]. The virus was subsequently found in Asia in sus monkey (Macaca mulatta) in 1947 in Uganda [13]. the 1960s. In 2007 there was an outbreak of ZIKV in Yap These strains were propagated and titrated to obtain the Island, Micronesia [14], which spread to Pacific islands 50% tissue culture infective dose per ml (­TCID50/ml) in in 2013 [15] before reaching Latin America in 2015 [16]. African green monkey kidney (VERO) cells. Nowadays, all isolated ZIKVs are grouped into three line- ages: Asian; African I; and West African II [17, 18]. ZIKV Experimental infection of mosquitoes has been isolated from numerous African mosquito spe- Forty-eight hours before exposure of mosquitoes to the cies in field [19] but Aedes aegypti is considered to be infectious blood meal, the 10% sucrose solution was the main vector of ZIKV in urban areas [20]. In addi- removed to increase mosquito appetite. Totals of 480 tion, several Aedes species from all continents have been Ae. caspius and 275 Ae. aegypti females (7–10 days old) observed to transmit the virus experimentally: e.g. Aedes were exposed to infectious blood at 7 ­log10 ­TCDI50/ml of vittatus, Aedes vexans [20], Aedes polynesiensis [21] and both Suriname and MR766 strains and 20 females of each Ae. albopictus [22–26]. species were exposed to non-infectious blood. For blood- Due to the ZIKV epidemics in the American continent, meal preparation, rabbit blood was washed and mixed some viraemic travellers coming from endemic coun- with adenosine 5’-triphosphate (ATP) (5 × 10−3 M) tries have been reported in Europe [27], especially dur- (Sigma-Aldrich, St. Louis, MO, USA) and virus (infec- Hemotek® artificial feed- ing the summer months [28], raising important alarms tious) or cell culture medium (DMEM) (non-infectious). for human health. In this context, more in-depth knowl- Females were exposed to a ­ edge is required about the susceptibility of autochtho- ing system (Discovery Workshop, Accrington, UK) at nous mosquito species such as Ae. caspius for ZIKV to 37.5 ± 0.5 °C for 30 min. After exposure, females were Núñez et al. Parasites Vectors (2019) 12:363 Page 3 of 7 extracted from the samples using ­NucleoSpin® RNA anesthetized with C ­ O2. Fully engorged females were Hilden, Germany) at 30 Hz for 1 min. Viral RNA was selected and maintained in groups of ten in cardboard cages (Watkins & Doncaster, Leominster, UK) under the Virus (Macherey-Nagel, Düren, Germany) according same rearing environmental conditions. Throughout the to the manufacturer’s protocol. Zika RNA was detected experiment, the mosquitoes were maintained with per- by reverse-transcription quantitative PCR (RT-qPCR) previously [32] and AgPath-ID™ One-Step RT-PCR manent access to 10% sucrose solution in cotton pledges. using the primers ZIKA 1086 and ZIKA 1162c defined After a period of 7, 14 and 21 days post-exposure (dpe) to infectious blood, females were anesthetized with C­ O2, reagents (Applied Biosystems, Foster City, CA, USA). legs and wings were dissected and saliva was extracted The nucleic acids were detected with a Real-Time PCR using the capillary technique as previously described 7500 Fast System (Applied Biosystems) with the follow- [30]. The number of mosquitoes tested at each time ing amplification protocol: 45 °C for 10 min; 95 °C for point is summarized in Table 1. Body, leg and wing sam- 10 min; then 45 cycles at 95 °C for 15 s and at 60 °C for ples were stored in 0.5 ml of Dulbecco’s modified Eagle’s 45 s. The RT-qPCR sensibility was 0.451 T ­ CID50/reac- medium (DMEM) (Lonza Group AG, Basel, Switzerland) tion for detection of MR766 and 0.035 T ­ CID50/reaction and the saliva samples in 0.190 ml of DMEM at − 80 °C for Suriname ZIKV strains. until ZIKV detection. Infection (IR), disseminated infec- tion (DIR) and transmission rates (TR) were estimated to evaluate the vector competence [31]. The IR represents Results virus replication in the midgut epithelial cells. The DIR Feeding rates were 56% (280/500) and 77.45% (213/275) shows that the virus was able to cross the midgut barrier for Ae. caspius and Ae. aegypti, respectively. and reach the hemocoel. The TR shows that the virus was For the Ae. caspius population, ZIKV was detected by able to cross the salivary glands barrier. We also meas- RT-qPCR only in body samples for both virus strains ured the transmission efficiency (TE), which refers to the used in the present study (Fig. 1). As detailed in Table 2, rate of mosquitoes with infectious saliva among the total the infection rate (IR) in Ae. caspius for the Suriname mosquitoes assayed. The experimental infection was per- ZIKV strain was 3.33% and 10% at 14 and 21 days post- formed at IRTA, CReSA BLS3 facilities. exposure (dpe), respectively. For the MR766 ZIKV strain, the IR was only 3.33% at 7 dpe. In addition, both ZIKV detection ZIKV strains were unable to induce a disseminated Virus detection from leg, wing and body samples was car- infection and transmission in Ae. caspius. ried out using 1/10 and 1/100 dilutions in 96-well plates In our positive control (Ae. aegypti PAEA strain), containing a Vero cell monolayer. Saliva samples were infection was detected at 7 dpe for both ZIKV strains titrated directly in 96-well plates in a Vero cell monolayer. tested by RT-qPCR and cytopathic effect while dissemi- Vero cells were maintained with DMEM supplemented nation and transmission were only found for the Suri- with 2% FCS and 2% of penicillin/streptomycin/nysta- name ZIKV strain at 14 and 21 dpe (Table 1 and Fig. 1). tin (1000 U/ml, 10 mg/ml and 500 U/ml, respectively; The transmission efficiency (TE) of Ae. aegypti for the Sigma-Aldrich) and incubated for seven days at 37 °C and Suriname ZIKV strain based on the cytopathic effect 5% ­CO2 until cytopathic effect observation. in Vero cells was 15% (3/20) and 5% (1/20) at 14 and Prior to viral RNA extraction, the samples were 21 dpe, respectively. homogenized using a TissueLyser II (Qiagen GmbH, Table 1 Summary of assays Mosquito species ZIKV strain tested Titer of ZIKV ­( TCID50/ml) No. of mosquitoes tested per time point tested 7 dpe 14 dpe 21 dpe Total Ae. caspius Suriname 7 ­log10 ­TCID50/ml 30 30 30 90 MR766 7 ­log10 ­TCID50/ml 30 30 30 90 Negative control No virus – – 8 8 Ae. aegypti Suriname 7 ­log10 ­TCID50/ml 20 20 20 60 MR766 7 ­log10 ­TCID50/ml 20 20 19 59 Negative control No virus – – 11 11 Abbreviation: dpe, days post-exposure Núñez et al. Parasites Vectors (2019) 12:363 Page 4 of 7 Ae. caspius Ae. aegypti Inverted Cycle Threshold (CT) 20 Strain value 30 MR766 Suriname 40 e e e e e e e e e e e e e e e e e e dp dp dp dp dp dp dp dp dp dp dp dp dp dp dp dp dp dp 7 7 7 7 7 7 14 21 14 21 14 21 14 21 14 21 14 21 Body Legs & Wings Saliva Body Legs & Wings Saliva Fig. 1 Results of the RT-qPCR for both mosquito species tested. The CT values are blue for the Suriname strain and red for MR766 Discussion Aedes aegypti from PAEA (French Polynesia), selected The present study demonstrates that the assessed as a positive control, was able to transmit the Suriname population of Ae. caspius from Catalonia was unable strain (Asian lineage). Suriname ZIKV infection, dissemi- to transmit the Suriname and MR766 ZIKV strains, nation and transmission were observed at 14 and 21 dpe. belonging to the Asian and African I phylogenetic lin- These results are in agreement with other experiments eages, respectively. Our results indicate that the Ae. reported on Ae. aegypti vector competence for ZIKV [35, caspius population has a strong midgut escape bar- 36]. However, the MR766 strain (African I lineage; the rier (MEB) since at 7 dpe we found infected bodies of historical strain isolated in 1947 in Uganda [13]) was una- MR766 ZIKV strain by RT-qPCR as well as at 14 and ble to disseminate and be transmitted in this mosquito 21 dpe to the ZIKV Asian strain but neither the dissem- species. This ZIKV strain is an old strain that has suffered inated infection nor the transmission were detected at several passages in mice and cells from various laboratory any of the time points analyzed. These findings are in sources that we assume may have influenced the vector agreement with the hypothesis that the MEB can limit competence assays in both assayed mosquito species. virus dissemination from the midgut to the hemocoel Furthermore, the inefficient dissemination and transmis- or secondary organs as reported for other arbovirus- sion in Ae. aegypti (PAEA strain) exposed to the MR766 mosquito species combinations [33, 34]. As the viral ZIKV strain were in agreement with previous results load detected by RT-qPCR was low in infected bod- reported by Diagne et al. [37]. The differences observed ies (Fig. 1), we suggest that the virus is replicating at a between our results and other vector competence experi- very low level. Therefore, the virus would not be able to ments in which dissemination and transmission of the cross the MEB and disseminate through the mosquito MR766 ZIKV strain were reported for Ae. aegypti [26, hemocoel to reach the salivary glands making it unable 35, 36, 38, 39], could be explained by the genetic varia- to transmit the virus. In addition, these results are in bility of Ae. aegypti populations as mentioned by Diagne accordance with a recent study in which another Ae. et al. [37]. Furthermore, it is known that temperature caspius population was assayed for a ZIKV strain of the can influence vector competence as described for several Asian lineage (Puerto Rico, 2015) under constant envi- mosquito species infected with other arboviruses (e.g. ronmental conditions [8]. It should be noted that in our dengue virus (DENV), CHIKV [40] or WNV [41–43]). A experiment, apart from testing a ZIKV strain of Asian recent study showed that temperature may directly affect lineage, we also assayed a ZIKV strain of the African I vector competence for ZIKV rendering the Ae. aegypti lineage (MR766) with cycled environmental conditions population tested at low temperature (18 °C) unable (26/22 °C day/night). Both studies indicate that despite to transmit the virus [44]. Therefore, the environmen- the anthropophilic behavior of Ae. caspius, its role in tal conditions used in the present study could have also the transmission of ZIKV seems unlikely. influenced our results; in earlier studies where higher Núñez et al. Parasites Vectors (2019) 12:363 Table 2 Infection, disseminated infection and transmission rates of Ae. caspius from Catalonia and Ae. aegypti PAEA populations artificially fed with Suriname and MR766 ZIKV strains at a titer of 7 ­log10 ­TCID50/ml. Viral detection was performed by the visualization of cytopathic effect on cells and RT-qPCR assays Dpe Species Suriname MR766 IR (%) DIR (%) TR (%) IR (%) DIR (%) TR (%) CE RT-qPCR CE RT-qPCR CE RT-qPCR CE RT-qPCR CE RT-qPCR CE RT-qPCR 7 Ae. caspius 0/30 (0) 0/30 (0) 0/0 (0) 0/0 (0) 0/0 (0) 0/0 (0) 0/30 (0) 1/30 (3.33) 0/0 (0) 0/1 (0) 0/0 (0) 0/0 (0) Ae. aegypti 17/20 (85) 20/20 (100) 0/17 (0) 0/20 (0) 0/0 (0) 0/0 (0) 2/20 (10) 2/20 (10) 0/2 (0) 0/2 (0) 0/0 (0) 0/0 (0) 14 Ae. caspius 0/30 (0) 1/30 (3.33) 0/0 (0) 0/1 (0) 0/0 (0) 0/0 (0) 0/30 (0) 0/30 (0) 0/0 (0) 0/0 (0) 0/0 (0) 0/0 (0) Ae. aegypti 20/20 (100) 20/20 (100) 9/20 (45) 19/20 (95) 3/9 (33.3) 8/19 (52.6) 2/20 (10) 2/20 (10) 0/2 (0) 0/2 (0) 0/0 (0) 0/0 (0) 21 Ae. caspius 0/30 (0) 3/30 (10) 0/0 (0) 0/3 (0) 0/0 (0) 0/0 (0) 0/30 (0) 0/30 (0) 0/0 (0) 0/0 (0) 0/0 (0) 0/0 (0) Ae. aegypti 19/20 (95) 19/20 (95) 16/19 (84.2) 19/19 (100) 1/16 (6.2) 17/19 (89.4) 1/19 (5.2) 1/19 (5.2) 0/1 (0) 0/1 (0) 0/0 (0) 0/0 (0) Abbreviations: CE, cytopathic effect; IR, infection rate; DIR, disseminated infection rate; TR, transmission rate; dpe, days post-exposure Note: Data are given as number of positive/number of tested samples (percentage) Page 5 of 7 Núñez et al. Parasites Vectors (2019) 12:363 Page 6 of 7 dissemination and transmission were observed in the Ae. Funding This study was funded by CERCA Programme/Generalitat de Catalunya. and aegypti-MR766 ZIKV pairing, the assays were performed the European Union’s Horizon 2020 Research and Innovation Programme at a temperature of 28 °C in contrast to the 26/22 °C (day/ under ZIKAlliance Grant Agreement no. 734548. night) used in our experiment (which we used to mimic Availability of data and materials summer environmental conditions in the area where the All data generated or analyzed during this study are included in this published Ae. caspius population was captured). article. Finally, with respect to the techniques used for virus Ethics approval and consent to participate detection in our vector competence assays, the RT- Not applicable. qPCR results had a slightly better sensitivity than those obtained by cytopathic effect in all mosquito-virus pair- Consent for publication Not applicable. ings. However, although RT-qPCR was more sensitive, the cytopathic effect caused by the virus allows better Competing interests knowledge of its viability which is more useful for a bet- The authors declare that they have no competing interests. ter estimation of the transmission efficiency of a mos- Author details quito population. Therefore, we strongly recommend 1 Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Centre de Recerca en that for the vector competence studies viable viruses in Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona, Bel‑ laterra (Cerdanyola del Vallès), 08193 Barcelona, Spain. 2 Servei de Control de the saliva should be taken in to account to determine the Mosquits, Consell Comarcal del Baix Llobregat, Barcelona, Spain. 3 Department transmission efficacy. of Virology, Arboviruses and Insect Vectors Unit, Institut Pasteur, Paris, France. Received: 10 January 2019 Accepted: 19 July 2019 Conclusions Given the high risk of ZIKV introduction in Europe via infected travelers coming from endemic areas, it is References important to know if anthropophilic European mosquito 1. Robert V, Günay F, Le Goff G, Boussès P, Sulesco T, Khalin A, et al. 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N Engl Europe. Euro Surveill. 2017;22:30347. J Med. 2016;375:1002–4. 30. Dubrulle M, Mousson L, Moutailier S, Vazeille M, Failloux AB. Chikungunya virus and Aedes mosquitoes: saliva is infectious as soon as two days after Publisher’s Note oral infection. PLoS ONE. 2009;4:e5895. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ 31. Talavera S, Birnberg L, Nuñez AI, Muñoz-Muñoz F, Vázquez A, Busquets N. lished maps and institutional affiliations. Culex flavivirus infection in a Culex pipiens mosquito colony and its effects Ready to submit your research ? 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