González et al. Parasites Vectors (2019) 12:150 https://doi.org/10.1186/s13071-019-3359-1 Parasites & Vectors RESEARCH Open Access Limited risk of Zika virus transmission by five Aedes albopictus populations from Spain Mikel A. González1,2, Márcio G. Pavan2, Rosilainy S. Fernandes2, Núria Busquets3, Mariana R. David2, Ricardo Lourenço‑Oliveira2,4, Ana L. García‑Pérez1 and Rafael Maciel‑de‑Freitas2,4* Abstract Background: Aedes albopictus, the Asian tiger mosquito, is an exotic invasive species in Europe. It has substantial public health relevance due to its potential role in transmitting several human pathogens. Out of the European coun‑ tries, Spain has one of the highest risk levels of autochthonous arbovirus transmission due to both the high density of Ae. albopictus and the extensive tourist influx from vector-endemic areas. This study aims to investigate the suscep‑ tibility of five Ae. albopictus populations from mainland Spain and the Balearic Islands to a Brazilian Zika virus (ZIKV) strain. Methods: The F1 generation of each Ae. albopictus population was orally challenged with a ZIKV-infected blood meal (1.8 × 106 PFU/ml). At 7 and 14 days post-infection (dpi), mosquito bodies (thorax and abdomen) and heads were individually analysed through RT-qPCR to determine the infection rate (IR) and dissemination rate (DR), respectively. The saliva of infected mosquitoes was inoculated in Vero cells and the transmission rate was assessed by plaque assay or RT-qPCR on ~33 individuals per population. Results: The IR and DR ranged between 12–88%, and 0–60%, respectively, suggesting that ZIKV is capable of cross‑ ing the midgut barrier. Remarkably, no infectious viral particle was found in saliva samples, indicating a low ability of ZIKV to overcome the salivary gland barrier. A subsequent assay revealed that a second non-infective blood meal 48 h after ZIKV exposure did not influence Ae. albopictus vector competence. Conclusions: The oral experimental ZIKV infections performed here indicate that Ae. albopictus from Spain become infected and disseminate the virus through the body but has a limited ability to transmit the Brazilian ZIKV strain through biting. Therefore, the results suggest a limited risk of autochthonous ZIKV transmission in Spain by Ae. albopictus. Keywords: Aedes albopictus, Zika virus, Spanish populations, Vector competence, Viral copies, RT-qPCR, Plaque assay Background trade of used tyres, lucky bamboo and flower pots, for Aedes albopictus (Diptera: Culicidae), also known as the instance [2]. So far, Ae. albopictus has been recorded in Asian tiger mosquito, is ranked as one of the world’s 100 27 European countries [3] since its introduction at the most invasive species in the world [1]. This species has end of the 1970s in Albania [4]. A few years later, this undergone a massive global expansion over recent dec- species became established in Italy, and by late 1990s ades facilitated by an effective network of air, ground and reached France [5], making its arrival to Spain immi- maritime human transportation, and the international nent at that time. Hence, in 2004, one larva in a tree hole and an adult male of Ae. albopictus were collected in the backyard of a house located near Barcelona [6]. *Correspondence: freitas@ioc.fiocruz.br In the following years, the presence of Ae. albopictus 2 Laboratório de Mosquitos Transmissores de Hematozoários, Instituto along the Mediterranean coast and Balearic Islands was Oswaldo Cruz, Fundação Oswaldo Cruz (IOC/ FIOCRUZ), Rio de Janeiro, Brazil also reported, with an upsurge in Catalonia and Valencia Full list of author information is available at the end of the article Autonomous Communities [7, 8]. © 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. González et al. Parasites Vectors (2019) 12:150 Page 2 of 9 The establishment of Ae. albopictus in Europe raises previously ZIKV-exposed females would affect the viral public health concerns since it is a competent vector of load in mosquito tissues. at least 26 arboviruses, including dengue (DENV), yel- low fever (YFV), chikungunya (CHIKV) and Zika (ZIKV) viruses [9]. Imported cases of CHIKV and ZIKV have Methods been reported in European countries infested by Ae. Mosquito populations albopictus, representing an alarming scenario that must Eggs of five Ae. albopictus populations from the Iberian not be overlooked [10, 11]. The role of Ae. albopictus as Peninsula and Balearic Islands, from Barcelona (BA), a vector of ZIKV has been confirmed by vector compe- Valencia (VA), Palma de Mallorca (PM), Málaga (MA) tence assays and by the detection of naturally infected and Guipúzcoa (GU) were collected with ∼12 ovitraps/ specimens [12]. locality placed on urban or peri-urban sites during the Since 2016, the European Union has reported about summer of 2017 (Fig. 1, Table 1). 2340 ZIKV-confirmed cases. Spain is the second most Field-collected ­F0 generation was reared to ­F1 adult affected country in the continent with 320 imported progeny to obtain enough mosquitoes to perform ZIKV cases, half of them reported in Catalonia [13], one of the oral infection experiments. Aedes albopictus females most likely port-of-entries for ZIKV in Europe due to were fed twice per week on human blood (CAAE its high touristic flow. Considering the high density of 53419815.9.0000.5248). Eggs were hatched, larvae were Ae. albopictus in Spain, especially in the Mediterranean reared at a maximum density of ≈500 individuals/3 region, it is essential to ascertain the vector competence l of water in plastic trays and fed daily with fish food of its local populations in order to estimate the potential (Tetramin, Tetra, Melle, Germany) until pupation. Pupae risk of autochthonous ZIKV transmission. were transferred to 30 × 30 × 30 cm cages (BugDorms, In this study, we assessed the ability of five Spanish Taichung, Taiwan) to allow adult emergence and mat- populations of Ae. albopictus to become infected, dis- ing. Colony mosquitoes were maintained in an insectary seminate and transmit a Brazilian ZIKV strain. We also at 27 ± 2 °C, 14:10 light:dark photoperiod, > 60% relative evaluated whether a second non-infected blood meal in humidity and supplied with 15% sucrose diet ad libitum. Fig. 1 Geographical distribution of Ae. albopictus and sampled populations in the Iberian Peninsula and Islands. The five sampling sites are denoted with stars. The current distribution of Ae. albopictus is adapted from Collantes et al. [7], ECDC [14] and Alcibar et al. [15]. Abbreviations: BA, Barcelona; VA, Valencia; PM, Palma de Mallorca; MA, Málaga; GU, Guipúzkoa González et al. Parasites Vectors (2019) 12:150 Page 3 of 9 Table 1 Mosquito populations and field-collected eggs related information from Spanish mainland and Balearic Islands Mosquito population Collection site Current status in ­Spaina Total no. of OPIc EDId ­eggsb Barcelona, Catalonia (BA) (41°48′33′′N, 2°03′33′′E) Established for a long time 500 88.8 96.4 Valencia, Valencian community (VA) (40°03′19′′N, 0°03′50′′E) Established for a long time 1300 77.7 185.8 Palma de Mallorca, Balearic Islands (PM) (39°41′45′′N, 2°42′0′′E) Recently established 760 83.3 76.1 Málaga, Andalusia (MA) (36°35′15′′N, 4°33′07′′W) Recently established 450 87.5 64.4 Guipúzcoa, Basque Country (GU) (43°20′35′′N, 1°45′44′′W) Occasionally introduced 54 4.1 9.1 a Established for a long time (> 7 years) and recently established (< 3 years) b Parental source of eggs (­ F0) c Ovitrap Positive Index (OPI): frequency of positive ovitraps among the total examined d Egg Density Index (EDI): no. of eggs / no. of positive ovitraps Virus strain performed after 24 h with 6–7 day-old female mos- The ZIKV strain (Asian lineage), namely ZIKV-PE243 quitoes using a membrane feeding system (Hemotek, (GenBank: KX197192), used in this study was isolated in Great Harwood, UK), adapted with a pig-gut covering. 2015 from a patient from Recife, Pernambuco (northeast The infective blood meal was offered in a proportion of Brazil), [16] where a substantial increase of microcephaly 1 ml of cell culture medium with virus to 2 ml of rab- cases in newborns was first detected in the Americas [17]. bit erythrocytes. Mosquito experimental infection was ZIKV was amplified in C6/36 mosquito cells (amplifica- done at a Biosafety Level-2 laboratory. Oral feeding was tion step < 10) maintained with Leibovitz’s L15 medium limited to 30 min and only fully engorged females were (Sigma-Aldrich, Missouri, USA) supplemented with 2% selected for vector competence assays. Mosquitoes fetal bovine serum (FBS, Gibco, Invitrogen, USA), and were held in cylinder plastic cups at 27 ± 0.5 °C, 12:12 h incubated for 7 days at 28 °C. Viral titers were quantified light:dark cycle with high relative humidity > 70%. Cot- through a serial 10-fold dilution via plaque-forming assay ton balls with sucrose solution (15%) were provided on in Vero cells (Sigma Aldrich, St. Louis, MO, USA) prior the top of the cups and replaced daily. to experimental infection, as reported by Fernandes et al. We analyzed 25 specimens of each of the five popula- [18]. The virus sample contained 1.8 × 106 PFU/ml and tions by RT-qPCR at 7 and 14 days post-infection (dpi) was stored at -80 °C until use. to determine the infection rate (IR) and dissemination rate (DR). The transmission rate (TR) was estimated by Experiment 1: Vector competence of five Ae. albopictus both plaque assay of saliva of 25 individuals at 7 and populations from Spain 14 days post-infection (dpi) per population and by RT- At 5–6 days post-emergence, about 500 females of each qPCR in 6–8 additional specimens per population at population were divided in groups of 65 specimens and 14 dpi (Table 2). transferred to cardboard feeding boxes without access to sucrose solution. Oral infection experiments were Table 2 Summary of ZIKV vector competence experiments of Ae. albopictus populations from Spanish mainland and Balearic Islands Mosquito No. of exposed Feeding rate (%) Positive infected Plaque ­assaya Total no. of samples RT-PCR population females females (%) analysed 14 dpib 7 dpi 14 dpi BA 500 52 88 25 25 150 8 VA 480 23 68 25 25 150 8 PM 392 26 24 25 25 150 6 MA 390 39 32 25 25 150 6 GU 442 39 84 25 25 150 8 a Plaque assay = 25 specimens per population (25 analysis of body, 25 analysis of head and 25 analysis of saliva) and condition b Number of saliva samples analysed Abbreviations: BA, Barcelona; VA, Valencia; PM, Palma de Mallorca; MA, Málaga; GU, Guipúzcoa González et al. Parasites Vectors (2019) 12:150 Page 4 of 9 Experiment 2: Effect of an additional uninfected blood with 45 µl of Leibovitz’s L15 medium supplemented with meal on the Ae. albopictus vector competence to ZIKV penicillin (10 μg/ml), gentamicin (1 μg/ml) and fungizone ZIKV-infected 6–7 day-old females were separated into (1 μg/ml). After 30 min of salivation, saliva samples and two groups: one-time blood-fed (1BF) and twice blood- mosquito bodies were immediately stored at -80 °C. fed (2BF). After 48 h (a proxy to simulate a second blood Vero cells were plated and incubated until monolayer ingestion on a natural setting), 2BF females were fed formation. Then, the entire sample of the saliva (50 μl) with an additional uninfected rabbit blood meal. Only for each mosquito was placed on a 6-well plate contain- 10 females from BA and 10 from GU fed twice from a ing the cells and incubated for 1 h (37 °C, 5% C­ O2) and total of 152 females (60 and 92, respectively). BA and GU supplemented with Earle’s 199 medium. After 1 h incu- were selected as representatives of different introduc- bation, the supernatant was removed, and the cells were tion histories in Spain, with the former established for overlaid with carboxymethyl cellulose (CMC) in Earle’s a long time and the latter occasionally introduced. The 199 medium. Inoculated Vero cells were incubated for viral RNA loads and rates were compared between 1BF 7 days and then fixed with 10% formaldehyde and stained and 2BF groups at 7 days after the first blood meal by with crystal violet (0.02%). To ensure cell viability and RT-qPCR. to prevent self-contamination during the process, 50 μl of 3-fold serial diluted ZIKV stock in Leibovitz’s L15 Sample processing and virus detection by RT‑qPCR medium was added in every assay to one of the plates as a The head and body (thorax and abdomen) of the mos- positive control and cell culture medium alone as a nega- quitoes stored at -80 °C were dissected on a chill-plate, tive control. and 250 μl of Leibovitz’s L15 medium supplemented with 4% FBS was added to vials filled with 20–25 glass beads. Viral quantification and statistical analysis Mosquito tissues were mechanically macerated using a The number of ZIKV-RNA copies in the body and head rapid homogenizer (Precellys 24, Bertin Technologies, was calculated with a ZIKV standard curve from 10-fold Montigny-le-Bretonneux, France) followed by centrifu- dilutions of known RNA copies (­101–109) included on gation for 10 min at 10,000× rpm and at 4 °C. Suspended every PCR plate. The limit of virus detection by RT-qPCR viral RNA was extracted from a 140 μl volume using was established in five RNA copies. IR and DR were cal- QIAamp Viral RNA Mini Kit (Qiagen, Dusseldorf, Ger- culated as the proportion of females with infected bod- many) following manufacturer’s instructions. ies among the total tested and the proportion of females The one step RT-qPCR amplification (­SuperscriptTM with infected heads among the total tested, respectively. III ­PlatinumTM, Invitrogen, Waltham, MA, USA) was TR refers to the total number of females with viral parti- performed as previously described [19] with some minor cles in saliva among the total tested. To ease comparison modifications. In brief, PCR reactions were performed with previous papers in which different mosquito popu- using 13.5 μl of master mix, 1.4 μl (10 μM) of each primer lations were challenged for ZIKV [18–22], we sought to (forward: 5′-CTT GGA GTG CTT GTG ATT-3′; and replicate the experimental design and estimate IR, DR reverse: 5′-CTC CTC CAG TGT TCA TTT-3′), 0.4 μl and TR in infected mosquitoes. Using the same ZIKV (50 mM) of ­MgSO4, 1.7 μl (10 μM) TaqMan TAMRA isolate described here, vector competence (demonstrated probe (FAM 5′-AGA AGA GAA TGA CCA CAA AGA by the presence of ZIKV particles in mosquito heads) TCA-3′ TAMRA), 0.4 μl of Taq polymerase, 5 μl of each ranged from 86–100%, highlighting good viral replication RNA sample and 1.2 μl of ultra-pure water to a final vol- and the reliability of the techniques used for viral detec- ume of 25 μl. Amplification reactions were performed in tion in varied tissues including mosquito saliva [21–23]. a QuantStudio 6 Flex Real-Time PCR System (Applied Fisher’s exact test was applied to detect significant dif- Biosystems, Foster City, CA, USA) programmed as fol- ferences between proportions (IR and DR). The number lows: 45 °C for 15 min and 95 °C for 2 min, followed by of viral copies was compared using the non-parametric 40 cycles of 95 °C for 15 s, 58 °C for 5 s and 60 °C for 30 s. Kruskall-Wallis test followed by pairwise comparisons. Mann-Whitney U-test was used to compare the viral Collection of saliva and virus detection by plaque assay titres between 7 and 14 dpi and between the groups of in Vero cells Experiment 2. Statistical analysis was performed with Female mosquitoes at 7 and 14 dpi (Experiment 1) and IBM SPSS statistics v.23.0. at 7 dpi (Experiment 2) were immobilised on a Petri dish placed on ice and their legs and wings were removed Results with forceps. The proboscis of each live mosquito was Indicators of Ae. albopictus infestation inserted into a 10 μl pipet tip containing 5 µl of FBS and A total 58 ovitraps were placed in the five regions from then expelled into a sterile 500 µl microcentrifuge tube Spain (Table 1). Of these, 75% had at least one Ae. González et al. Parasites Vectors (2019) 12:150 Page 5 of 9 albopictus egg. The ovitrap positive index (OPI) ranged (Fig. 2a). Remarkably, TR was zero for all populations as between 4–90% and the egg density index (EDI) ranged ZIKV particles were not detected in the saliva of any of between 9–96 in July 2017. All geographical locations the tested females by either plaque assay or RT-qPCR. showed support for a high level of infestation by Ae. albopictus mosquitoes, except for the GU population, which showed a low frequency of positive ovitraps and a Viral loads in mosquito tissues reduced number of eggs (Table 1). At 7 dpi, viral copies in bodies were similar among populations, but more ZIKV copies were found in Viral competence parameters mosquito bodies from BA, VA and GU than in mos- In total, 38% of the females exposed to the infective quitoes from PM and MA at 14 dpi (Kruskal-Wallis blood meal were fully engorged (Table 2). Overall, the test, W = 49.9, df = 4, P ≤ 0.001) (Fig. 2b). On the other IR and DR increased at 14 dpi, except for the MA pop- hand, the number of ZIKV copies in the heads at 7 dpi ulation for which a decrease was observed over time. was not statistically different among populations. The Overall, the IR ranged from 39.2 to 55.2% and the DR number of viral copies in the body was heterogeneous varied from 13 to 36% at 7 and 14 dpi, respectively. A at 14 dpi and specimens from PM were found with less high level of heterogeneity was observed in the IR and ZIKV copies in the body compared to the other popula- DR among populations. BA, VA and GU populations tions (Kruskal-Wallis test, W = 32.5, df = 4, P ≤ 0.0001) showed superior permissiveness than PM and MA (Fig. 2b). Overall, the number of ZIKV copies had a to ZIKV, with a higher DR at 7 dpi (Chi-square test, 19-fold increase in infected heads (Mann-Whitney test, χ2 = 13.8, df = 4, P = 0.008) and IR and DR at 14 dpi U = 1552.0, P = 0.207) and 13-fold in infected bodies (IR: χ2 = 48.8, df = 4, P ≤ 0.001 and DR: χ2 = 31.7, df = 4, (U = 216.0, P = 0.038) from 7 to 14 dpi. P ≤ 0.001) (Fig. 2a). On the other hand, mosquitoes from PM and MA presented low values of IR and DR. Effect of a second blood meal on vector competence No ZIKV-disseminated infection was observed in Ae. A second feeding on non-infected blood did not affect IR albopictus from VA at 7 dpi and from MA at 14 dpi and DR (Mann-Whitney test, BA IR: U = 54.00, P = 0.781; Fig. 2 Viral competence parameters in the five Spanish Ae. albopictus populations. Infection and dissemination rates (a) and number of ZIKV RNA copies (b). Infection rates and absolute number of ZIKV copies in both body and head of the five Ae. albopictus populations from Spain at days 7 and 14 after oral experimental exposition with a ZIKV infectious blood meal (1.8 × 106 PFU/ml). The five populations (BA, Barcelona; VA, Valencia; PM, Palma de Mallorca; MA, Málaga; GU, Guipúzcoa) are represented by different colours. Horizontal black bars represent the medians. The number of infected samples from the 25 mosquitoes analysed for each population are shown in parentheses. Different letters indicate statistically significant differences (Mann-Whitney U-test; ***P < 0.001) González et al. Parasites Vectors (2019) 12:150 Page 6 of 9 BA DR: U = 55.00, P = 0.654; GU IR: U = 42.50, P = 0.293; and egg density retrieved from our data, the infestation GU DR: U = 26.00, P = 0.482) or number of viral cop- index can be considered as high as in the regions where ies (BA body: U = 46.00, P = 0.796; BA head: U = 45.00, Ae. albopictus has been established for a long time or P = 0.739; GU body: U = 44.50, P = 0.684; and GU head: was recently introduced, which must concern public U = 43.00, P = 0.631) (Fig. 3). Although not statistically health managers. The exception is the Basque Country, significant, the mean ZIKV copies barely decreased in where Ae. albopictus eggs were occasionally detected in the BA population (1BF body: 2.3 × 105 ± 1.3 × 105; 2BF restricted places near the border with France. In that par- body: 3.0 × 104 ± 2.5 × 104; 1BF head: 78.4 ± 76.7; 2BF ticular case, public health authorities from the Basque head: 5.1 ± 3.9 for 2BF), while the GU population exhib- Country must enhance vector surveillance and control ited an opposite outcome (1BF body: 8.7 × 105 ± 7.0 × 105, on the border to avoid Ae. albopictus establishment. 2BF body: 3.7 × 106 ± 2.8 × 106; 1BF head: 34.4 ± 29.6; The vector competence of Ae. albopictus to ZIKV has 2BF head: 1.4 × 105 ± 1.1 × 105). Thus, an additional hitherto shown heterogeneous results, with the suscep- blood-feeding did not influence vector competence of tibility status of field populations oscillating dramati- Spanish Ae. albopictus to ZIKV. cally [12, 25, 26]. Our study has shown that Spanish Ae. albopictus populations are heterogeneously capable Discussion of becoming infected and disseminate ZIKV, but none Vector competence is a key feature to determine the risk were competent to transmit this virus strain through of pathogen transmission by mosquitoes. The success of biting. The same ZIKV strain used herein showed high a given mosquito population in transmitting a virus will replication rates and was detected in Ae. aegypti saliva, depend on the ability of the virus to overcome both the demonstrating a high transmission rate [21, 22]. On the midgut and salivary gland vector barriers [24]. Herein, European scale, Ae. albopictus from France, Italy and we report the susceptibility of recently introduced and Germany have demonstrated variable susceptibility long-established Ae. albopictus populations from Spain to Asiatic ZIKV lineages [10, 27–29]. Our findings are to ZIKV. These five populations represent the cur- aligned with those reported for Ae. albopictus popula- rent Ae. albopictus distribution in the country, portray- tions from France and Italy [10, 29], which showed only ing a nationwide sampling effort. Taken together, the few specimens with ZIKV in the saliva. Altogether, these results presented here suggested that this ZIKV strain results reinforce the hypothesis that Ae. albopictus from is not transmitted during female blood-feeding due to Mediterranean Europe might have limited vectorial the absence of infectious viral particles in Ae. albopictus capacity, at least for the ZIKV strain circulating in Bra- saliva at 7 and 14 dpi. zil. Nonetheless, conclusions regarding the absence of Aedes albopictus has colonized the majority of the ZIKV in Ae. albopictus saliva must be taken carefully for Spanish Mediterranean area since its arrival in 2004 [7] at least two reasons. First, forced salivation is a common and its geographical distribution is still expanding with method to diagnose arbovirus presence in saliva, but recent introductions in the Basque Country and Aragón its collection is obtained after mosquitoes are immobi- [14]. According to the frequency of positive ovitraps lized with legs and wings removed. Therefore, the use of Fig. 3 Comparison of ZIKV infection rates of Ae. albopictus populations exposed or non-exposed to an additional ZIKV non-infective blood meal. Infection rates (body and head) at 7 dpi of females that received a single ZIKV-infective blood meal (1BF) and those females fed on a second non-infective blood meal 48 h after viral exposure (2BF) are shown. Ten specimens were analysed for each condition. Populations: BA (Barcelona) and GU (Guipúzkoa). The numbers in parenthesis indicate the positive infected females González et al. Parasites Vectors (2019) 12:150 Page 7 of 9 non-sacrificial methods to collect saliva such as using a not show statistically significant differences in viral copies strip of filter paper, must be encouraged for vector com- after infected mosquitoes received a second non-infective petence studies [30]. Secondly, and most importantly, blood meal. Aedes albopictus females from the PV popu- considering a mosquito species a negligible vector might lation had a slightly increased viral load after the second produce undesired consequences such as the relaxation blood meal, while the opposite was observed for BA. of vector control. Amuzu et al. [37] evaluated the effect of multiple blood The viral concentration used in our study was slightly meals on dengue dynamics in Ae. aegypti and concluded lower compared to other studies conducted with Euro- that repeat feeding did not lead to an increase of IR or pean populations of Ae. albopictus [10, 29], but with viral loads. Interestingly, a second non-infective blood more viral particles than are naturally found in infected meal shortened the extrinsic incubation period (EIP) of hosts [12]. In addition, environmental factors such as ZIKV-infected Ae. albopictus [38]. Our data did not sup- temperature may affect vector competence. For instance, port the increased percentage of mosquitoes observed the susceptibility of Ae. albopictus for CHIKV is affected by Armstrong et al. [38] who offered the second blood depending on the incubation temperature of immature meal 4 days after ZIKV infection; in our study the second stages [31, 32]. Moreover, our infection was conducted blood meal was offered only 48 h after infection. Blood- with fresh-wild populations (F1 generation), which might feeding triggers mechanical and physiological changes in reflect better the vector competence of field populations mosquitoes that could affect virus replication and thus [33]. the EIP. Therefore, it is possible that the effects of a sec- Besides suitable environmental conditions, the estab- ond blood meal on virus replication is dependent on the lishment of an arbovirus transmission cycle depends on time after infection it is offered to mosquitoes [39]. the genetic background of mosquito populations and the Furthermore, the vector potential of other species to viral genotype [34, 35]. In Spain, two main hypotheses transmit ZIKV in Spain should also be considered. The of Ae. albopictus introduction have been proposed, but recently sporadic incursion of Ae. aegypti supported by not confirmed. The first is the dispersion along the entire maritime transport represents a recent rising concern in Spanish Mediterranean coast including the Balearic Spain. In December 2017, Ae. aegypti mosquitoes were Islands since their introduction and establishment in reported in the Spanish outermost region of the Canary Barcelona. The second possible way of arrival is by occa- Islands, a popular destination for travellers [40]. Indeed, sional introductions from France to Guipúzkoa (north- the new arrival and or/establishment of other exotic mos- east Spain) [7, 8]. In this scenario, mosquitoes from quito species of Aedes, i.e. Ae. japonicus, in a rural area of the Guipúzkoa population may proceed from a non- north Spain, suggests that the Spanish epidemic potential established population, while the Barcelona and Valen- to arboviruses is dynamic and could change underlying cia females might come from a long-established one. the rapid global spread of invasive species [41]. Despite that, GU, BA and VA exhibited higher IR and DR, whereas Palma de Mallorca and Málaga populations (collected in areas recently colonized by Ae. albopictus) Conclusions showed an overall lower virus infection and dissemina- The results from the oral experimental infections car- tion. This lower vector competence could be explained by ried out in the present study suggest that Spanish Ae. an impairment between mosquito and virus genotypes. albopictus populations are capable of becoming infected The validation of this hypothesis depends on increasing and disseminating ZIKV to other tissues. However, Ae. the number of tested samples, future research on popu- albopictus seems to have a limited ability for transmitting lation genetics of these mosquitoes and assessing the the ZIKV strain used herein since no viral particles were vector competence of local Ae. albopictus for different detected in saliva. Additional vector competence assays strains of ZIKV but also other arboviruses. Until then, with other ZIKV strains must be conducted to increase vector surveillance authorities must remain alert to pro- the confidence of appointing Spanish Ae. albopictus as a duce a rapid response and effective vector control when non-competent vector. suitable. Aedes albopictus may feed on blood more than once in Abbreviations a single gonotrophic cycle [36]. Hence, understanding if CHIKV: chikungunya virus; CMC: carboxymethyl cellulose; CT: cycle threshold; repeated blood-feeding improves the viral load and infec- FBS: fetal bovine serum; DENV: dengue virus; dpi: days post-infection; DR: dissemination rate; EDI: egg density index; EIP: extrinsic incubation period; IR: tion rates is relevant from an epidemiological point of infection rate; OPI: ovitrap positive index; PFU: plaque-forming unit; PCR: poly‑ view, as the risk of arbovirus transmission might increase merase chain reaction; RT-qPCR: reverse transcription qPCR; TR: transmission with repeated contact with hosts. Although the results of rate; YFV: yellow fever virus; ZIKV: Zika virus. Experiment 2 were disparate between populations, it did González et al. Parasites Vectors (2019) 12:150 Page 8 of 9 Acknowledgements 5. Schaffner F, Karch S. Premiere observation d’Aedes albopictus (Skuse, The authors thank the entomologists who kindly donated tiger mosquito 1894) en France metropolitaine. Compt Rend Academie Sci Ser III. eggs from different parts of Spain: Mikel Bengoa, Javier Lucientes, Sarah Dela‑ 2000;323:373–5. cour, Francisco Collantes and Joseba Goikolea. We are also grateful to Fiocruz 6. Aranda C, Eritja R, Roiz D. First record and establishment of the mosquito staff for technical support and laboratory assistance. Aedes albopictus in Spain. Med Vet Entomol. 2006;20:150–2. 7. Collantes F, Delacour S, Delgado JA, Bengoa M, Torrell-Sorio A, Guinea H, Funding et al. Updating the known distribution of Aedes albopictus (Skuse, 1894) in Funding was supported by FAPERJ E-26/201.335/2016. MAG receives a 3-year Spain 2015. Acta Trop. 2016;164:64–8. postdoctoral fellowship funded by the Basque Government. Coordenação de 8. Eritja R, Palmer JRB, Roiz D, Sanpera-Calbet I, Bartumeus F. Direct evidence Aperfeiçoamento de Pessoal de Nível Superior (Grant No. 23038.007199/2012- of adult Aedes albopictus dispersal by car. Sci Rep. 2017;7:14399. 17 and Finance Code 001). 9. Paupy C, Delatte H, Bagny L, Corbel V, Fontenille D. Aedes albopictus, an arbovirus vector: from the darkness to the light. Microbes Infect. Availability of data and materials 2009;11:1177–85. The data supporting the conclusions of this article are included within the 10. Jupille H, Seixas G, Mousson L, Sousa CA, Failloux AB. Zika virus, a new article. Raw data are available from the corresponding author upon request. threat for Europe? PLoS Negl Trop Dis. 2016;10:e0004901. Samples are temporally stored at -80 °C in the Laboratório de Mosquitos 11. Marano G, Pupella S, Pati I, Masiello F, Franchini M, Vaglio S, et al. Ten years Transmissores de Hematozoários (IOC/ FIOCRUZ). since the last chikungunya virus outbreak in Italy: history repeats itself. Blood Transfus. 2017;15:489–90. Authors’ contributions 12. Epelboin Y, Talaga S, Epelboin L, Dusfour I. Zika virus: an updated review Experimental conception and design: MGP, RLO and RMF. Vector competence of competent or naturally infected mosquitoes. PLoS Negl Trop Dis. experiments: MAG, MGP, RSF and MRD. Analysis and interpretation: MAG, MGP 2017;11:e0005933. and RMF. Paper writing: MAG, RMF, RLO, NB and ALG. All authors read and 13. ECDC. European Centre for Disease Prevetion and Control. Zika transmis‑ approved the final manuscript. sion; 2018. https​://ecdc.europ​a.eu/en/zika-virus​-infec​tion/threa​ts-and- outbr​eaks/zika-trans​missi​on. Accessed 10 July 2018. Ethics approval and consent to participate 14. ECDC. European Centre for Disease Prevetion and Control. Aedes albop- Mosquito blood-feeding was performed with human blood obtained from ictus - current known distribution in Europe; 2018. https​://ecdc.europ​ anonymous donors from the blood bank of the Rio de Janeiro State University, a.eu/en/disea​se-vecto​rs/surve​illan​ce-and-disea​se-data/mosqu​ito-maps. whose blood bags would have been discarded due to small volume. We Accessed 10 July 2018. have no information on donors, including sex, age and clinical condition. The 15. Alcibar RM, Tello A, Marino Hernando E, Vázquez Martinez MA. Aedes use of human blood was approved by the Fiocruz Ethical Committee (CAAE (Stegomyia) albopictus (Skuse, 1894) (Diptera, Culicidae) primera cita para 53419815.9.0000.5248). la Comunidad de Madrid, España. Boln Asoc Esp Ent. 2017;41:3–4 (In Spanish). Consent for publication 16. Faria NR, Azevedo RDSDS, Kraemer MUG, Souza R, Cunha MS, Hill SC, et al. Not applicable. Zika virus in the Americas: early epidemiological and genetic findings. Science. 2016;352:345–9. Competing interests 17. Lowe R, Barcellos C, Brasil P, Cruz OG, Honório NA, Kuper H, et al. The The authors declare that they have no competing interests. Zika virus epidemic in Brazil: from discovery to future implications. Int J Environ Res Public Health. 2018;15:E96. 18. Fernandes RS, Campos SS, Ferreira-de-Brito A, Miranda MR, Silva KAB, Publisher’s Note Gonçalves MC, et al. Culex quinquefasciatus from Rio de Janeiro is Springer Nature remains neutral with regard to jurisdictional claims in pub‑ not competent to transmit the local Zika virus. PLoS Negl Trop Dis. lished maps and institutional affiliations. 2016;10:e0004993. 19. Ferreira-de-Brito A, Ribeiro RM, Miranda RM, Fernandes RS, Campos SS, Author details Silva KA, et al. First detection of natural infection of Aedes aegypti with 1 NEIKER-Instituto Vasco de Investigación y Desarrollo Agrario, Derio, Bizkaia, Zika virus in Brazil and throughout South America. Mem Inst Oswaldo Spain. 2 Laboratório de Mosquitos Transmissores de Hematozoários, Instituto Cruz. 2016;111:655–8. Oswaldo Cruz, Fundação Oswaldo Cruz (IOC/ FIOCRUZ), Rio de Janeiro, Brazil. 20. Chouin-Carneiro T, Vega-Rua A, Vazeille M, Yebakima A, Girod R, Goindin 3 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la D, et al. Differential susceptibilities of Aedes aegypti and Aedes albopictus Universitat Autònoma de Barcelona, Bellaterra, Spain. 4 Instituto Nacional de from the Americas to Zika virus. PLoS Negl Trop Dis. 2016;10:e0004543. Ciência e Tecnologia em Entomologia Molecular, Universidade Federal do Rio 21. Fernandes RS, Campos SS, Ribeiro OS, Raphael LM, Bonaldo MC, de Janeiro, Rio de Janeiro, Brazil. Lourenço-de-Oliveira R. 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PLoS Negl Trop Dis. 2016;10:e0005101. resident species and on human health. Ecol Lett. 2005;8:558–74. 34. Ciota AT, Bialosuknia SM, Zink SD, Brecher M, Ehrbar DJ, Morrissette MN, et al. Effects of Zika virus strain and Aedes mosquito species on vector competence. Emerg Infect Dis. 2017;23:1110–7. Ready to submit your research ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations • maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions