Assessment of Culex pipiens bioforms in the world’s southernmost distribution limit

The mosquito Culex pipiens s.s. L. occurs as two bioforms that differ in physiology and behaviour affecting virus transmission cycles. To assess the occurrence of Cx. pipiens bioforms in the southernmost limit of its distribution, specimens were collected aboveground in southern Buenos Aires Province and east Patagonia, Argentina. Ten larvae and 25 adults were individually processed and identified by polymerase chain reaction (PCR) amplification of Ace-2 and CQ11 loci. Culex quinquefasciatus Say (one larva, two adults), Cx. pipiens f. molestus (one larva, one adult) and one adult of hybrid origin were identified in Buenos Aires Province; only Cx. pipiens f. molestus was recorded in Patagonia (eight larvae, 21 adults). The potential absence of bioform pipiens and its implications in arbovirus enzootic cycles is discussed.

These ecophysiological differences have implications in disease transmission, increased by the fact that hybrids show opportunistic feeding behaviour and may therefore serve as bridge vectors for virus transmission from infected birds to humans or other dead-end hosts. (10) The aim of this work was to address for the first time the presence of Cx. pipiens bioforms in the world's southern extreme of its distribution. Sampling was conducted from southern Buenos Aires Province to east Patagonia region along the Atlantic coast ( Figure), under the hypothesis that bioform molestus is present in the northern extreme of the study area and is gradually replaced by bioform pipiens in the southern end. Buenos Aires Province is characterised by cumulative precipitation values between 500 and 1,100 mm west-east and a mean annual temperature gradient ranging from 13.4ºC southeast to 17.9ºC northwest; (11) isotherms present a non-flat pattern as the result of the joint interaction of topography and oceanic influence. Argentinean Patagonia extends over the southern cone of South America, limited by the Colorado River to the north, the Atlantic Ocean to the east and the Andean cordillera to the west (Figure). It includes a wide variety of soils, climatic conditions and vegetation formations; mean annual temperatures range from 16ºC in the northwest to 3ºC in the south. (11) The eastern portion of the territory is drier than the west (< 250 mm per year), as the zonal atmospheric flow and transport of humid air from the Pacific Ocean are blocked by the Andes Cordillera. The arid steppe is intersected by four main rivers, which margins concentrate most human settlements. All specimens of Cx. pipiens previously collected throughout Patagonia have been identified by morphological or enzymatic techniques; therefore, no bioform differentiation is available. (12,13) Specimens of Cx. pipiens s.l. were collected during March 2018 and January 2019 along and in the vicinities of the key communication route between southern Buenos Aires and east Patagonia, covering over 1700 km from 36.36 to 47.18ºS (Figure). Collections were performed in cemeteries, tire repair shops, river margins, and public toilets of fuel stations or other facilities. Adult mosquitoes were caught with manual and battery-powered hand aspirators whereas immatures were collected by dipping in discarded vehicle tires, uncovered water tanks and flower vases. All specimens were preserved at -16ºC in a portable car freezer until arrival to the laboratory. There, DNA from individual specimens (either larvae or adults) was extracted with one of the following methods: approximately 50% were individually ground with sterilised mortar and pestle and genomic DNA was extracted using the EasyPure Genomic DNA extraction kit (Transgen Biotech), whereas DNA from the other 50% was extracted with 4 M ammonium hydroxide following Vogels et al. (14) The second inexpensive method was used for sampling locations with many specimens, whereas silica extraction columns were reserved for scarce samples (one or two individuals at a given location).
Specimen identification followed the polymerase chain reaction (PCR) protocols by Smith & Fonseca (15) and Bahnck & Fonseca (16) for the amplification of the second intron of the Ace-2 nuclear gene and the 5′ flanking region of microsatellite locus CQ11, respectively. The first protocol amplifies a 610-bp band for Cx. pipiens and a 274-bp band for Cx. quinquefasciatus [Supplementary data (Figure)], whereas the simultaneous presence of both bands is indicative of hybrid signatures. The second distinguishes between both forms of Cx. pipiens amplifying a 250-bp band for molestus and a 190-200-bp band for pipiens; again, the presence of both bands indicates hybrid signatures. As a result of cross-reactivity of primers, Cx. quinquefasciatus amplifies a PCR product similar in size to that of Cx. pipiens f. molestus in the CQ11protocol [Supplementary data ( Figure)]; therefore, the combined use of both is mandatory in sympatric areas. A negative (distilled water) control and positive controls from Cx. pipiens f. pipiens (Southern France), Cx. pipiens f. molestus (England) and Cx. quinquefasciatus (Indonesia) were included in all runs. A 5 μL aliquot of each amplified product mixed with 1 μL of loading buffer 6x was electrophoresed in a 2% agarose gel containing ethidium bromide (0.5 μg/ mL) and 0.5X TBE buffer. Bands were visualised under Location letters correspond to those in Figure. In column ID (for identification), P, Q and H indicate Cx. pipiens f. molestus, Cx. quinquefasciatus and the hybrid between the two, respectively; the number of identified specimens is reported between brackets (n). a gel UV transilluminator. DNA ladders of 50 and 100 bp precision were run in parallel to allow size estimation of observed bands. Amplified PCR product of the southernmost collected specimen was purified with DNA PuriPrep-GP kit (INBIO Highway) and sequenced in an ABI 3130xl Genetic Analyzer (Applied Biosystems) by a third-party provider. Sequences were edited using ApE v2.0.55 and compared to known sequences by a BLAST search comparison with the GenBank DNA database (www.ncbi.nlm.nih.gov/blast/Blast.cgi).
We collected specimens of Cx. pipiens s.l. from Tapalqué (Buenos Aires Province, 36.36ºS) to Comodoro Rivadavia (Chubut Province, 45.87ºS) in cemeteries (two positive / four inspected), tire repair shops (5/10), river margins (2/5) and public toilets (8/32) (Table). Only adults or larvae were collected at any given positive site with the exception of a cemetery (site 28), in which immatures were collected in a flower vase and one female was aspirated from the vegetation surrounding the same grave.
Adult collections were performed mainly in public toilets of fuel stations (sites 1, 2, 11, 21, 22, 24, 25 and 33) or cemeteries (site 41); in one tire repair shop, all tires were dry but adults were resting inside its cavities (site 6). A large number of adult specimens were recorded in humid environments associated with river margins; at a camping site (site 12), plenty of adults were collected beneath grill spots and inside rubber dustbins, whereas at a canal near the river (site 35) adults were found resting under a tree near the ground. Larval collections were performed in vehicle tires at sites 4, 10, 13 and 18 (Table). South of Comodoro Rivadavia (location P in Figure), we inspected four public toilets, one tire repair shop, one cemetery and one river margin but no adults were found and all potential larval habitats were dry (details in Table footnote).
A total of 10 larvae and 25 adults were individually processed; all DNA extractions using both methods resulted in successful PCR identifications. Specimens were identified either as Cx. quinquefasciatus, Cx. pip-Occurrence of the members of the Culex pipiens complex in southeast Argentina. Letters indicate sampling locations, see Table for details. iens f. molestus or hybrids between the two, remarking the absence of Cx. pipiens f. pipiens [Supplementary data ( Figure)]. Both Cx. quinquefasciatus (one larva, one female, one male), Cx. pipiens f. molestus (one larva, one female) and a hybrid Cx. quinquefasciatus/Cx. pipiens f. molestus (one female) were collected in Buenos Aires Province, whereas Cx. pipiens f. molestus was the only member of the complex recorded throughout east Patagonia (eight larvae, 15 females, six males). Top blast matches of the sequence of the CQ11 amplified PCR product of the female collected at Comodoro Rivadavia were Cx. pipiens f. molestus (100% identity and query cover with reference specimen DQ470149 (16) ).
The results reported herein represent first-to-date molecular identifications of Cx. pipiens bioforms in its world's southernmost distribution. Although in small numbers, we collected both larvae and adults of the complex in different land uses, and all specimens of Cx. pipiens s.s. were consistently identified as form molestus. Previous studies in Buenos Aires Province identified specimens from La Plata City (34.87ºS, 57.90ºW) as form molestus by a full microsatellite analysis, (17) whereas the individuals collected in 13 cemeteries throughout the province were also identified as molestus using the CQ11 locus. (18) Although no previous attempts to identify Cx. pipiens bioforms had been made in Patagonia, Mitchell et al. (19) reported a high degree of autogeny and proof of anthropophily in a colony established from mosquitoes collected at 43ºS. This constitutes a worthy of note precedent of the occurrence of form molestus in the distribution fringe of Cx. pipiens, and the potential absence of form pipiens territory wide. Although the molestus form was originally considered to be limited to belowground and confined breeding sites, during the past decade it has been reported to occur naturally in open and aboveground habitats, even at high latitudes in the Northern Hemisphere with more extreme climatic conditions than those recorded in Patagonia (e.g. 14 ). Our current finding in Comodoro Rivadavia is the southernmost record of Cx. pipiens f. molestus in South America.
Argentinean members of the Cx. pipiens complex were reported to be competent but only moderately efficient vectors and less susceptible to WNV than comparable U.S. mosquito strains. (17) Although no attempt to isolate WNV or SLEV from wild avifauna or mosquitoes have been conducted in Patagonia, these arboviruses have been reported throughout North America at latitudes similar or even higher than the ones in to the southern counterpart. (2,7) If the bioform pipiens is actually absent, other Culex species could be responsible for the maintenance of arbovirus enzootic cycles. Alternatively, in Patagonia Cx. pipiens f. molestus could show opportunistic behaviour and feed on birds. In North America, at the beginning of the season Cx. pipiens has been reported to feed on birds (particularly the American robin Turdus migratorius), whereas after bird migration it switches to human feeding, therefore acting as an excellent bridge vector. (20) No host preference studies of the bioforms have been yet conducted in South America, highlighting an information gap to be fulfilled by future studies together with the aim of drawing a more complete distribution map of the bioforms in southern Argentina.
Our biotyping method assigns specimens into one of three possible discrete outcomes (molestus, pipiens or their hybrids) based on a single locus. Although this assay is considered a reliable diagnostic method, we acknowledge that the genetic structure of the Cx. pipiens bioforms is far more complex. To shed light on this matter and gain insight in the evolutionary relationships of the South American strain, selected specimens were recently included in a whole genome sequencing project currently under development, along with other specimens from all around the global range of the species.