Characterization and Host-Feeding Patterns of Culex pipiens s.l. Taxa in a West Nile Virus-Endemic Area in Southeastern Romania

Abstract

Culex pipiens sensu lato has been documented as West Nile virus (WNV) vector in southeastern Romania. Bucharest, the densely populated capital city of Romania, and the surrounding Ilfov county are WNV hotspots. In this area, the morphologically indistinguishable biotypes of Cx. pipiens, namely pipiens and molestus, are usually differentiated by their behavioral and physiological traits. Their involvement in WNV transmission, as suggested by entomological investigations, was not previously documented for each biotype. We used a Real-Time PCR assay based on CQ11 microsatellite to identify the Cx. pipiens biotypes and their hybrids collected in various habitats in the Bucharest metropolitan area. A sympatric distribution of both biotypes was observed, with a preference of green areas for pipiens, and human settings and animal farmlands for molestus. In the latter habitats, pipiens and molestus were found in mixed aboveground populations. A low number of hybrids was found suggesting existence of reproductive isolation. In subway tunnels molestus was dominant with a higher number of hybrids recorded than aboveground. Blood-engorged mosquitoes were identified to biotype and the blood meal source identified by DNA barcoding. Overall, Cx. pipiens s.l. fed mainly on birds, commonly on house sparrows, collared doves, and blackbirds, which are potential WNV-amplifying hosts. The preference for avian hosts was expressed strongest by pipiens biotype, while molestus was substantially less specific, feeding on avian and mammal hosts with similar frequency, with humans representing 20% of the hosts. Hybrids had a host choice closer to that of molestus. These findings highlight the role of pipiens biotype as enzootic/epizootic vector, and specifically show molestus as the bridge vector for WNV. The pipiens and molestus biotypes show important differences in habitat preferences, including oviposition; these findings demonstrate that targeted mosquito control to limit WNV transmission may be possible.

Introduction

The role of hematophagous arthropods in the transmission cycle of vector-borne infectious diseases is well established. Mosquitoes are vectors for important viral diseases such as yellow fever, dengue, chikungunya, Zika, and West Nile fever (Napp et al. 2018). Culex pipiens sensu lato mosquitoes have been identified as the main vectors of West Nile virus (WNV) in southeastern Romania (Savage et al. 1999, Dinu et al. 2015). This is the area that encompasses the highly populated Bucharest capital city. The area is endemically affected by WNV since the mid-1990s, with significant outbreaks in humans in the years 1996, 2010, and 2016–2019 (Tsai et al. 1998, Sirbu et al. 2011, Popescu et al. 2018, ECDC 2019). Furthermore, this area is susceptible to the introduction of WNV; to date strains of both lineage 1 and 2 of WNV have been detected. Different strains of lineage 2 cocirculated and thereafter replaced each other (Cotar et al. 2018). According to the weekly reports of Romanian National Institute of Public Health during the recent WNV outbreaks of 2016–2019, 124 WNV encephalitis cases were recorded in the Bucharest metropolitan area, of which 21 were lethal.

In Europe Culex pipiens complex (Cx. pipiens s.l.) is represented only by Cx. pipiens species with its two biotypes, pipiens and molestus. Culex torrentium Martini, considered a sibling species of Cx. pipiens, is also occurring (Becker et al. 2012, Harbach 2012).

In Romania, a country with temperate climate, the two biotypes of Cx. pipiens and Cx. torrentium were recorded. In this study, the presence of Cx. torrentium was excluded based on the entomological observation and molecular diagnosis (Prioteasa and Cotar, unpublished data). The presence of both molestus and pipiens biotypes in urban settings in Romania was first reported in the 1980s. The two biotypes were at that time differentiated by underground larval habitat and autogeny for molestus, and aboveground habitat and anautogeny for pipiens (Dancescu et al. 1980). There are additional behavioral and physiological traits which differentiate the two biotypes, including mating habits, feeding preference, oviposition, and overwintering (Vinogradova 2000). The pipiens biotype females are ornithophilic, eurygamous, anautogenous, and overwinter in a state of diapause (Zittra et al. 2016). On the other hand, molestus females are stenogamous, mammalophilic, and strongly anthropophilic, frequently biting indoors (endophilic), autogenous, and do not diapause (Kent et al. 2007, Gomes et al. 2013). These two biotypes are geographically separated in northern parts of Europe, and sympatric in southern Europe. The pipiens biotype prefers the aboveground habitat while the molestus biotype is mainly found in underground habitats, such as the ones found in the human-made urban environment (Martinez-de la Puente et al. 2016), however, in southern Europe both biotypes may share aboveground habitats (Yurchenko et al. 2020). The involvement of molestus biotype in the transmission of WNV has been speculated following the entomological investigation of the first human outbreak of WNV in Bucharest in 1996 (Savage et al. 1999).

There is no complete reproductive isolation between the two biotypes, leading to the emergence of hybrids that can be identified only by molecular techniques (Kothera et al. 2010). Considering the different behavioral characteristics among the parental biotypes and their hybrids, each could potentially impact WNV circulation and transmission to humans differently (Vogels et al. 2017). The investigation of host-feeding preferences through blood meal analysis is important in determining the role of each biotype in the transmission cycle of WNV in a given area (Munnoz et al. 2011, Osorio et al. 2012, Hernandez-Triana et al. 2017).

This study focused on establishing the structure of the Cx. pipiens complex in a WNV-endemic area in Romania, the distribution of molestus and pipiens biotypes in various habitats in the region, and the identification of host-feeding preference for the biotypes in the understanding of the ecology of WNV in an urbanized area of southeastern Europe.

Materials and Methods

Study area

The study area (1821 km²) is located in southeastern Romania, in the Romanian Plain, at an altitude that ranges between 50 and 120 meters above sea level. It comprises the Bucharest metropolitan area, with the urban Bucharest — capital city of Romania — with a population of 2,151,665 inhabitants (NIS 2020a) and surrounding Ilfov county with a population of 451,839 people (NIS 2020b). Bucharest is crossed by the Dâmbovița river, and is a mosaic of administrative, new business areas, old neighborhoods of individual dwellings with gardens, intermingled with neighborhoods of blocks of flats, parks, a chain of lakes, and wetlands. The Văcărești Natural Park, a wetland within the Bucharest city limits, was one of the mosquito sampling sites in this study. The park is on the site of an abandoned hydrotechnical project 4 km away from the city center, and covers more than 1.83 km². The vertebrate fauna is mainly represented by birds, of roughly 138 observed species, whereas mammals are poorly represented (VNP 2020). Ilfov county used to be a rural agricultural area in the 1990s. Urbanization has brought the county closer to the city, with many new residential areas and businesses, keeping its traditional dwellings, some farmland and patches of plain forest. Both the capital Bucharest and Ilfov county are affected yearly by WNV outbreaks resulting in severe impact on public health.

Mosquito collection and identification

Mosquito specimens were selected among the collections conducted during mosquito seasons of years 2011–2019 for the study of WNV transmission. These include samples from various habitats, therefore at least 50 specimens per type of habitat were selected for analysis, together with all the engorged females (Table 1). Aboveground habitats, including downtown administrative centers, residential neighborhoods with blocks of flats, individual households, and green areas such as parks and wetlands, as well as underground habitats such as the subway tunnels were sampled within the city limits. Farmlands plus their premises, and residential areas with individual dwellings were sampled in the surrounding Ilfov county. Centers for Disease Control and Prevention (CDC) gravid traps (John W. Hock Company, Gainesville, FL) and hand aspirators (Hausherr's Machine Works, Toms River, NJ) were used in various habitats within Bucharest and surrounding Ilfov county. CDC light traps (John W. Hock Company, Gainesville, FL) and hand aspirators were used to collect mosquitoes in subway tunnels. No specific permits were necessary to conduct the field studies. Mosquitoes were morphologically identified using the keys of Becker (Becker et al. 2010). Samples were kept at −70°C until processing for biotype identification and blood meal analysis.

Table 1.

The Distribution of Culex pipiens Biotypes in Various Habitats from Bucharest Metropolitan Area, 2011–2019

Cx. pipiens biotype based on CQ11 locus	Bucharest city						Ilfov county				Total for Bucharest metropolitan area
	Aboveground				Underground		Aboveground
	Downtown plus residential neighborhoods		Green area wetland		Subway tunnels and hallways		Traditional dwellings and residential		Farm and premises
	n	%	n	%	n	%	n	%	n	%	n	%
Pipiens	79	46	56	98	1	2	57	67	17	22	210	47
Molestus	78	45	1	2	45	80	24	28	58	74	206	46
Hybrids	15	9	0	0	10	18	4	5	3	4	32	7
Total	172	100	57	100	56	100	85	100	78	100	448	100

Biotype identification

Each blood-engorged mosquito was individually dissected and its abdomen removed under stereo microscope for blood meal analysis. The rest of the material was kept for biotype identification. DNA was extracted from abdomen and legs using a commercial kit (ReliaPrep™ Blood gDNA Miniprep System, Promega) following the manufacturer's instructions. The two biotypes of Cx. pipiens and their hybrids were identified using a multiplex real-time PCR assay based on CQ11 locus as described elsewhere (Rudolf et al. 2013).

Blood meal analysis

Blood meal analysis was based on a DNA barcoding protocol described elsewhere (Alcaide et al. 2009) with few modifications. Briefly, the first round of PCR amplification was carried out in a final volume of 50 μL using GoTaq^® Green Master Mix (Promega) and primers M13BCV-FW and BCV-RV1 at a concentration of 0.4 μM each. Ten microliters of DNA prepared as described above were used as template. Five microliters of round one PCR mix was used for the nested PCR with primers M13 and BCV-RV2 at a concentration of 0.4 μM each. Amplicons were sequenced with the BigDye™ Terminator v3.1 Cycle Sequencing Kit on a 3130 Genetic Analyzer (Applied Biosystems) and the resulting sequences were visually inspected and manually edited with BioEdit version 7.2.5 (Hall 1999). Cytochrome c oxidase subunit I (COI) sequences were analyzed using BOLDSYSTEMS (Ratnasingham et al. 2007).

Results

Culex pipiens s.l. mosquitoes were collected at 16 sites, nine located in Bucharest city and seven in Ilfov county, respectively (Fig. 1). The biotype was assigned for a total number of 448 specimens as follows: 210 (47%) pipiens, 206 (46%) molestus, and 32 (7%) hybrids. The distribution of Cx. pipiens biotypes in different habitats sampled in Bucharest metropolitan area is presented in Table 1. The results show a sympatric distribution of the two biotypes and their hybrids in the study area. However, there was clear variation in biotype distribution in different habitats, both within the city limits and in the periurban areas. The molestus biotype was found underground in the subway tunnels in high proportions (80%), and was accompanied by molestus × pipiens hybrids (18%). In the green areas of the city, the pipiens biotype represented 98%, molestus being rarely found. However, both biotypes were collected in similar numbers in the residential neighborhoods and downtown administrative areas, and with the proportion of hybrids being low (9%). Pipiens biotype was more frequently collected in traditional dwellings and residential areas (67%) in periurban settings than molestus, the latter being dominant in animal farmland premises (74%).

FIG. 1.

Location of the 16 mosquito sampling sites in Bucharest city and Ilfov county, southeastern Romania, 2011–2019.

Of 448 Cx. pipiens s.l. mosquitoes analyzed, only 217 were visibly blood engorged. Less than 1% (2) of engorged mosquitoes were collected in April–May, 9% (21) in June, 21% (45) in July, 49% (106) in August, and 20% (43) in September. The composition of collection of engorged mosquitoes followed the population dynamics with a peak in August. Most of the blood-engorged mosquitoes were collected with the CDC Gravid traps, not in shelters, and belonged to both biotypes.

Samples that produced quality sequences (216) with satisfactory match of 98–100% in BOLDSYSTEMS were further considered and included in the final analysis. No mixed blood meals were detected. Vertebrate partial COI gene was successfully sequenced in 54 blood meals from Cx. pipiens biotype pipiens, 113 from Cx. pipiens biotype molestus, 15 from pipiens x molestus hybrids, and for 34 individuals of Cx. pipiens s.l. (biotype not determined) (Table 2). A total of 21 host species were identified as 17 avian and four mammalian species, including humans. Birds dominated the host species list (63%, 136 blood meals), whereas mammals represented only 37% of the hosts (80 blood meals). The most common avian host species were: the house sparrow (25.5%, 55 blood meals), followed by collared dove (13.4%, 29 blood meals), blackbird (7.4%, 16 blood meals), and chicken (7.4%, 16 blood meals), followed by other species (9.3%, 20 blood meals) with two or one blood meal(s). The most common mammalian host species was human (15.3%, 33 blood meals), followed by canine (10.2%, 22 blood meals), bovine (9.3%, 20 blood meals), and feline (2.3%, 5 blood meals) (Table 3).

Table 2.

Blood Meal Distribution for Cx. pipiens Biotypes

	Pipiens biotype n/frequency (%)	Molestus biotype n/frequency (%)	Hybrids n/frequency (%)	N/A ^a biotype n/frequency (%)	Total Cx. pipiens s.l. n/frequency (%)
Birds	44/82	58/51	6/40	28/82	136/63
Non-human mammals	5/9	33/29	4/27	5/15	47/22
Humans	5/9	22/20	5/33	1/3	33/15
Total	54/100	113/100	15/100	34/100	216/100

Not tested due to the financial constraints.

Table 3.

Source of Mosquito Blood Meals Analyzed in This Study

Blood meal source	Pipiens biotype	Molestus biotype	Hybrids	N/A ^a biotype	Total
Birds	44 (82%)	58 (51%)	6 (40%)	28 (82%)	136 (63%)
Accipiter brevipes (levant sparrowhawk)		1			1
Apus apus (common swift)	1				1
Columba livia (var. domestica, feral pigeon)	4	2		1	7
Corvus cornix (hooded crow)			1		1
Corvus corone (carrion crow)	2				2
Gallus gallus (var. domesticus, chicken)	1	12		3	16
Larus cachinnans (caspian gull)				1	1
Luscinia luscinia (thrush nightingale)			1		1
Numida meleagris (var. domestica, guineafowl)		1			1
Nycticorax nycticorax (black-crowned night heron)		1			1
Parus major (great tit)	1				1
Passer domesticus (house sparrow)	17	26	2	10	55
Pica pica (Eurasian/common magpie)		1			1
Saxicola rubetra (whinchat)			1		1
Streptopelia decaocto (Eurasian collared dove)	9	6	1	13	29
Sturnus vulgaris (common starling)	1				1
Turdus merula (Eurasian/common blackbird)	8	8			16
Mammals	10 (18%)	55 (49%)	9 (60%)	6 (18%)	80 (37%)
Non-human mammals	5 (9%)	33 (29%)	4 (27%)	5 (15%)	47 (22%)
Bos taurus (bovine)	1	19			20
Canis lupus familiaris (dog)	2	12	3	5	22
Felis catus (cat)	2	2	1		5
Homo sapiens (human)	5 (9%)	22 (20%)	5 (33%)	1 (3%)	33 (15%)
Total blood meals determined	54 (100%)	113 (100%)	15 (100%)	34 (100%)	216 (100%)

Not tested due to the financial constraints.

Of the pipiens biotype 44 (82%) out of 54 mosquitoes collected fed on birds, 5 (9%) on non-human mammals, and 5 (9%) on humans. Of the molestus biotype 58 (51%) out of 113 individuals fed on birds, 33 (29%) on non-human mammals, and 22 (20%) on humans. In the 15 hybrids tested, six fed on birds, four on non-human mammals, and five on humans (Table 3).

Discussion

We present data about the structure of Cx. pipiens complex and host choice of pipiens and molestus biotypes, as well as of their hybrids, in a WNV endemic urban/periurban habitat of a highly populated area in southeastern Romania. While pipiens biotype is highly dominant in green areas, both pipiens and molestus biotypes share the aboveground habitats in the residential and administrative public areas of the city with similar frequency. Molestus was found in the subway tunnels and hallways as previously documented (Byrne et al. 1999). The urban habitat offers a variety of larval habitats, resting sites, and sanctuaries to overwinter without diapause. The artificial containers filled with water, the sewage system, the urban water catchment basins, and the wastewater treatment plants found in this environment represent the perfect water sources necessary for larval development (Ruiz et al. 2007). Towns are considered heat islands in which adult Culex mosquitoes emerge earlier than in other habitats, and these factors increase the risk of vector-borne infections (Townroe et al. 2014). In periurban areas molestus biotype was found in higher numbers than pipiens biotype especially in farmland areas, perhaps due to the wastewater infrastructure in animal farms. Hybrids were found in low numbers aboveground, both in the city and periurban area, ranging from 4% to 9%, suggesting that despite the sympatric distribution, some reproductive isolation mechanisms exist. Low proportion of hybrids was reported in Germany (1.7–6.6%), where molestus biotype was also low (Rudolf et al. 2013). In the Mediterranean countries, the proportion of hybrids was higher for Portugal (17.5%) (Osorio et al. 2012) and West of Spain (25.7%), pipiens being found as the dominating biotype (66%) for the latter (Bravo-Barriga et al. 2017). A high proportion of hybrids (36.8%) associated with high numbers of molestus biotype (48.3%) was reported in Algeria (Amara Korba et al. 2016) and thought to be associated with the harsh Saharan environment. These reports suggest that the proportion of a certain biotype is not the main trigger of hybridization, but other factors may be involved; in fact when one biotype is in low frequency, the occurrence of hybrids indicate a significant hybridization rate.

In this study, hybrids were found aboveground in areas in which molestus/pipiens ratios varied from 0.42 to 3.4. However, in subway tunnels the ratio of molestus/pipiens was 45 and higher number of hybrids was found (10 out of 56; 18%), suggesting that the low number of pipiens biotype mosquitoes getting underground produce hybrids at an increased rate. A plausible explanation for these results may be that molestus males swarm in small spaces close to the ground, and may potentially mate with pipiens females that accidentally get underground, whereas aboveground pipiens males swarm at 2–3 meters high, close to tree canopy isolated from molestus females (Yurchenko et al. 2020).

Generally, molestus biotype prefers to feed on mammals and rest in confined spaces such as underground garages or basements, whereas pipiens biotype prefers to feed on avian hosts and rest in open-air habitats, being epigeous (Di Luca et al. 2016). Our host preference study performed on 54 individuals of pipiens biotype, 113 molestus and 15 hybrids confirmed in part these findings. Indeed, pipiens fed mainly on birds (82%), and much less on mammals, including humans. However, molestus biotype showed almost an even preference for bird host (51%) and mammal host species (49%) with 20% of all blood meals being from human host. Although the number of tested hybrids was low, their host choice seemed similar to that of molestus with preference for mammals, and more so for humans, suggesting that hybridization skews the host preference of hybrids to that of the molestus biotype parent.

The majority of the vertebrate host were avian species (n = 17) and only a few (n = 4) were mammalian species (Table 3). The most frequent avian host identified in our study was house sparrow (40% of all avian blood meals), a species with competence as amplifying host for WNV (Del Amo et al. 2014). Taking into account that house sparrows are the most abundant passerine population in the area, it is possible that the species is selected by mosquitoes opportunistically as being easily available. A preference of Cx. pipiens s.l. mosquitoes for house sparrows, blackbirds, and collared doves as blood-feeding hosts in a WNV hotspot in northern Italy was also reported by (Rizzoli et al. 2015). Collared doves seem to have a low competence as WNV reservoir (Panella et al. 2013), but could compensate this by population abundance. As for the European blackbird there are no data, but other species of genus Turdus from the New World proved to be competent amplifying hosts acting as a WNV super spreader (Kilpatrick et al. 2006).

In urban conditions, the biodiversity is low and this is reflected also by the host choice found in mosquito populations. In the studied region, green areas are intricate with administrative and residential areas, and there was a certain diversity of bird species, some of which are WNV amplification hosts, also.

Our data address main aspects of the transmission cycle of WNV in the area, bird species acting as amplifying hosts, and mosquitoes being involved in enzootic/epizootic transmission as well as in transmission to humans as bridge vectors. Since 1996, WNV has caused outbreaks among people and was detected in Cx. pipiens s.l. mosquitoes in the study area (Savage et al. 1999, Sirbu et al. 2011, Dinu et al. 2015, Cotar et al. 2018). Vertical transmission of WNV has also been documented for Cx. pipiens s.l. in the region (Dinu et al. 2015) and may represent a significant mechanism of virus maintenance. In earlier studies on WNV in southeastern Romania, it was observed that virus circulation in birds may be intense as revealed by seroprevalence studies, without transmission to humans, and pipiens biotype might be the vector sustaining it (Ceianu et al. 2001). In this study, molestus fed evenly on birds and mammals, humans representing 20% of molestus blood meal host. This host preference, together with its abundance and endophilic behavior, suggest molestus biotype as the main bridge vector responsible for WNV transmission to humans. Such a vector role has already been suggested in other studies (Vogels et al. 2017).

These findings have significant implications for vector CDC of WNV outbreaks. The current vector control strategy in the area is based on the use of chemical adulticides often performed late in the season, as a reaction to WNV outbreaks and have low impact on transmissions. Only in urban natural wetland reserve control measures target larval stages, using environment-friendly bacterial larvicides. Our data show that molestus may be a key determinant of WNV transmission to humans suggesting that mosquito control should target habitat reduction, where it is feasible, or reduce its preadult population in its typical hypogean larval habitats. Flooded basements, cellars, rain drains with sediment traps, and water in subway tunnels, represent habitats for molestus larvae in Bucharest city (Prioteasa, unpublished).

Conclusions

This is the first study on the structure of Cx. pipiens complex in southeastern Romania, a hotspot area for WNV. Our findings show that the two biotypes, molestus and pipiens, occur in a sympatric distribution with low numbers of hybrids. Furthermore, host preference investigation suggests pipiens biotype to act as enzootic/epizootic vector, and molestus biotype to play a significant role as bridge vector of WNV.

Footnotes

Authors' Contributions

F.L.P. and E.F. performed field collection, entomological study, and prepared the specimens for molecular tests. G.V.T., I.G.S., and A.I.C. performed molecular identification of biotype. A.I.C., G.V.T., I.G.S., E.F., and S.D. performed blood meal analysis. G.V.T., I.G.S., S.D., F.L.P., E.F., C.S.C., and A.I.C. drafted the article. S.D., A.I.C., and C.S.C. revised the article. C.S.C. coordinated the study.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This study was supported from the budget of the following national grants of the Romanian Ministry of Research and Innovation: PN-II-PT-PCCA-2013-4-0718, grant 126/2014; Core Program PN 16 39 01 01, grant 46N/2016; CCCDI-UEFISCDI project number PN-III-P1.2-PCCDI-2017-0005/2018, within PNCD III; and Core Program PN 19 14 02 04, grant 47N/2019. The laboratory methods were established in the framework of European Union (EU) grant FP7-261504 EDENext.

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