Integrated Approaches in Support of Taxonomic Identification of Mosquitoes (Diptera: Culicidae) in Vector Surveillance in Spain

Collection and identification of specimens

Mosquitoes were collected between 2016 and 2018 in 19 localities in La Rioja and in 1 wetland nearby in Navarra region (northern central Spain) (Fig. 1). Mosquito specimens used in this study were sampled with several collecting techniques such as BG-Sentinel traps, human landing catch, collecting adults in resting places, ovitraps, and sampling immature stages by dipping following the methodology detailed in Ruiz-Arrondo et al. (2019). Morphological identifications were based on descriptions given in the identification keys of Schaffner et al. (2001) and Becker et al. (2010). In the case of eggs collected in ovitraps, which did not hatch and develop to adults, DNA was extracted directly from them without morphological identification.

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FIG. 1.

Collection localities in La Rioja. 1: Pantano de La Grajera (Logroño); 2: Iregua River (Logroño); 3: Embalse de Las Cañas (Viana); 4: Laguna de La Degollada (Calahorra); 5: Laguna de Hervías (Hervías); 6: Venta de Alfaro (Alfaro); 7: Laguna de la Estanca (Calahorra); 8: Alhama River (Alfaro); 9: Plaza Toros (Haro); 10: Portilla River (Mansilla); 11: Najerilla River (Mahave); 12: Linares River (Cervera del río Alhama); 13: Irrigation channel (Valdegutur); 14: Añamaza River (Valdegutur); 15: Laguna del Recuenco (Calahorra); 16: Pantano del Perdiguero (Calahorra); 17: Ebro River (Arrubal); 18: Oja River (Ezcaray); 19: Lagunas de Rabanera (Ajamil); 20: Sheep farm (Ortigosa de Cameros). The map was created using QGIS 2.8.

DNA extraction and PCR

DNA was extracted from mosquito legs following the protocols of Hernández-Triana et al. (2017, 2019). In the case of eggs collected in ovitraps, some eggs from the same wooden stick were pooled, and DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). PCR primers were those developed by Folmer et al. (1994) (LCO1498 and HCO2190), which are considered the standard for the amplification of the 658 bp region of the COI gene (Hebert et al. 2003a,b). In addition, we also used the primers 5.8SF and 28SR of Collins and Paskewitz (1996) for the amplification of the ITS2 gene. The COI PCR was carried out for species belonging to both Culicinae and Anophelinae, whereas the ITS2 PCR was only carried out in species within Anophelinae as it offers a good species delineation within this subfamily (Linton et al. 2005).

PCR products were obtained using the profile conditions and reaction mix in Hernández-Triana et al. (2017, 2019). All PCR products were visualized on a 1.5% agarose gel, and samples showing bands of the correct size were sequenced in both directions using the ABI PRISM^® BigDye^® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). All sequences were assigned to a particular mosquito species when agreement was ≥98% with sequences of named species in the GenBank.

Detailed specimen records and sequence information (including traces files) were uploaded to the Barcode of Life Data Systems (BOLD, www.boldsystems.org). The Digital Object Identifier (DOI) for the publicly available data set is as follows: “dx-doi-org.web.bisu.edu.cn/10.5883/DS-MQSVLR”; dx-doi-org.web.bisu.edu.cn/10.5883/DS-MLRJ. All sequences have been submitted to GenBank: COI-accession numbers: MK402666 to MK402927, MN413913 to MN413939; and ITS2-accession number: MK412721 to MK412767. All voucher specimens are housed at the Animal and Plant Health Agency, United Kingdom and the Center for Biomedical Research of La Rioja (CIBIR), Spain.

Sequence analysis

Paired bidirectional sequence traces for COI or ITS2 were combined to produce a single consensus sequence. All sequences were analyzed in MEGA v.6 (Tamura et al. 2013). Neighbor-Joining (NJ) analysis was carried out using the K2P distance metric to represent their clustering pattern; bootstrap values were calculated to test the robustness of the phenogram and were obtained by conducting 1000 pseudoreplicates (Hernández-Triana et al. 2019). A Barcode Index Number (BIN) was assigned to sequences longer than 500 bp, and each BIN was mapped according to species (Table 1). The taxonomic discordance in the dataset was analyzed using BOLD as detailed in Ratnasingham and Hebert (2013).

COI sequence analysis

In total, 465 COI DNA barcode sequences were analyzed. All individuals morphologically identified as the same species clustered together and no deep splits in the NJ tree were observed (Fig. 2). Our dataset was found to match to 23 BINs. In general, all barcode sequences were assigned a BIN number, which represented 99% concordant BINs, one BIN was a singleton (Ae. berlandi, BOLD: ACY5176). The only single discordance between the BINs in our dataset was found in a single specimen identified as Culex (Cx.) torrentium, which appears to share a BIN within Culex pipiens (BOLD: AAA4751; Process ID: MSEMV855-15).

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FIG. 2.

Neighbor-joining tree of COI DNA barcodes (658 bp) for mosquito species. A divergence of >2% may indicate separate operational taxonomic units. Only bootstrap values higher than 70% are shown. COI, cytochrome c oxidase subunit I.

In the Anopheles (An.) maculipennis complex, all individuals were identified as Anopheles atroparvus, and COI distinguished Anopheles plumbeus and Anopheles algeriensis. In the case of the members of Anopheles claviger complex, An. claviger s.s. and Anopheles petragnani no COI barcode sequences were available in public data bases. Thus, our material was identified in this study as An. claviger s.l. (Fig. 2), which separated well from the other species of Anopheles.

All species of genus Aedes and Culex were successfully resolved in the NJ tree (Fig. 2). However, COI did not separate the forms of Cx. pipiens group (forms pipiens and molestus).

Our DNA barcode dataset for the genus Culiseta separated species such as Culiseta (Cs.) annulata, Culiseta litorea, Culiseta longiareolata, and Culiseta subochrea with high-support bootstrap values (Fig. 2). However, certain specimens were morphologically misidentified due to variation identified in the main morphological characters used to identify these species. During this study, three specimens identified as Cs. annulata in the NJ tree with the typical crosswing veins pattern of Cs. annulata (Fig. 4D) were detected, but the abdominal scales were yellow as in Cs. subochrea (Fig. 4G). In addition, four specimens grouped in the NJ tree as Cs. subochrea had the r-m and r-cu veins closer (Fig. 4H), similar to the pattern found in Cs. annulata (Fig. 4D).

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FIG. 4.

Typical morphological characters to distinguish species in the genus Culiseta, and morphological variations found in this study. Typical female abdominal coloration pattern of: (A) Culiseta annulata; (B) Culiseta subochrea; (C) Culiseta litorea. Typical female wing venation of: (D) Cs. annulata; (E) Cs. subochrea. (F) Male genitalia of Cs. litorea. Morphological variations: (G) Female with wing venation of Cs. annulata and yellow abdominal scales as in Cs. subochrea. (H) Female with abdominal coloration pattern of Cs. subochrea with crosswing venation as in Cs. annulata. (I) Female of Cs. litorea with indistinct V-shaped pattern in abdominal sternites. (J) Female fore leg coloration pattern of Cs. litorea; inset, higher magnification of tarsomeres I–V. Arrows indicate key morphological characters.

The typically inverted V-shaped pattern in the abdominal sternites of female specimens of adult Cs. litorea often was not distinguishable due to the loss of the scales (Fig. 4I). In addition, the basal pale ring of the tarsomere IV of the hind leg was difficult to distinguish in most specimens (Fig. 4J). These features can cause confusion during identification of Culiseta morsitans, if the structure is damaged. DNA barcodes from nine male specimens with typical characters of Cs. litorea grouped together in the same cluster where females were tentatively identified as Cs. litorea and Cs. morsitans; therefore, these females were judged to be Cs. litorea.

The levels of sequence divergence were variable across the taxa, with conspecific individuals collected from a single site often exhibiting 0, or 0.11% to 2.62% divergence values for species with more than three specimens (Table 1). The average intraspecific genetic divergence measured 1.30%.

The interspecific divergence ranged between 3.05% and 20.07% (Appendix Table A1). Interspecific genetic divergence values were higher between species from different genera. The pairs An. plumbeus/Coquillettidia richiardii (20.07%) and An. algeriensis/Cq. richiardii (20.04%) were the most divergent species. The smallest values of interspecific genetic divergence were found among species in the same genus, for example, Cx. pipiens/Cx. torrentium (3.05%), Cs. annulata/Cs. subochrea (3.58%), and Cx. torrentium/Culex theileri (6.05%) (Appendix Table A1). Culex impudicus was the only species with higher levels of divergence above 2% (2.62%) (Table 1).

ITS2 sequence analysis

In total, 54 ITS2 sequences of Anophelinae were obtained. All individuals morphologically identified as the same species clustered together and there were not deep splits in the NJ tree (Fig. 3). However, we were unable to obtain complete ITS2 sequences from our specimens of Anopheles claviger s.l. to be included in the dataset, because of incomplete amplification. Instead, ITS2 sequences available in the NCBI database were used for both species of the complex: An. claviger s.s. (Germany, Italy and Tajikistan) and An. petragnani (Germany), although only a single ITS2 sequence was available for the latter species. The intraspecific genetic divergence ranged from 0% to 2.98%, and was above 2% in An. claviger s.s. (table not shown).

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FIG. 3.

Neighbor-joining tree of ITS2 sequences (475 bp) for Anopheles species collected in La Rioja, Spain. A divergence of >2% may indicate of separate operational taxonomic units. Only bootstrap values higher than 70% are shown. ITS2, internal transcribed spacer 2.

Genetic diversity in the subfamily Anophelinae

For certain species of Anopheles, COI is not a reliable genetic marker for species identification, for example, Anopheles messeae and Anopheles daciae (Hernández-Triana et al. 2019); however in our dataset An. maculipennis s.l. separated well from other members of the genus Anopheles. The high genetic diversity for An. claviger s.l. (2.69%) identified in the COI analysis is indicative of the presence of both species of the complex in the studied area as pointed previously by Bueno-Marí (2012) for La Rioja. Nevertheless, we were unable to detail which species of the An. claviger s.l. inhabits the wetlands sampled because no longer fragments of the ITS2 gene for our individuals of An. claviger s.l. were obtained. For this reason, sequences of both species from the NCBI database were used, and ITS2 separated well An. claviger s.s. from An. petragnani (Fig. 3).

As a result of a paucity of molecular data for both species in public databases, we advocate for studies to further obtain genetic data for these species in Europe.

The use of ITS2 allowed us to identify An. atroparvus as the only member of the An. maculipennis s.l. present in the sampled localities.

No evidence of genetic diversity in certain species within the genera Culex and Aedes

No ambiguous identification was detected in specimens identified as Ae. cantans in relation to Ae. annulipes, when sequences were compared in NCBI. Lilja et al. (2018) and Hernández-Triana et al. (2019) reported some genetic differentiation within the population of Ae. vexans, but no deep divisions were shown in the COI NJ tree, and the genetic diversity was below 2% (Table 1). This might be due to the close proximity of the localities we sampled; therefore, further collecting is needed to delineate Ae. vexans populations in the Iberian Peninsula. Cx. impudicus was the only species with a genetic divergence above 2%, although this was not reflected as a deep split in the NJ tree (Fig. 2) and BOLD assigned the same BIN (AAB6945) to the 10 individuals morphologically identified as Cx. impudicus.

With regard to Aedes subgenus Finlaya, the genetic diversity was 0.71% for Ae. geniculatus clade, where a single record (a cluster of eggs not hatched collected in ovitraps) from La Rioja is included together with 1 specimen available in GenBank from the United Kingdom, and 15 specimens from Belgium. Despite BOLD assigned the same BIN (AAM5898) to all sequences of Ae. geniculatus, our specimen identified as Ae. geniculatus differed between 3.06% and 3.98% from the Ae. geniculatus clade in BOLD (3.82–3.98% in GenBank), and 5.05% from the only sequence of Aedes echinus in GenBank.

These findings closely resemble the values reported by Krüger et al. (2014) for specimens from South Germany. Adults of Ae. geniculatus are difficult to separate from Ae. echinus and Aedes gilcolladoi. Larvae of Ae. geniculatus and Ae. echinus are distinguishable morphologically, but no samples were taken from tree holes, the larval habitat of these species. Hence, our findings corroborate the possible existence of sibling species in Ae. geniculatus and merits further investigations. Only a single COI sequence of Ae. echinus was available in GenBank and no sequences for Ae. gilcolladoi was present, as a result, we are unable to comment further on the status of the three European Aedes (Finlaya) species.

COI reveals taxonomic problems between species in the genus Culiseta

COI DNA barcoding highlighted misidentifications within the genus Culiseta. Current morphological keys to identify European mosquitoes (Becker et al. 2010) distinguish the females of Cs. annulata and Cs. subochrea by the differing coloration and the distribution of the scales on the terga, the relative position of the crosswing veins r-m and m-cu, and the presence of dark and pale scales on costa (C), subcostal (Sc), radius (R) and cubitus (Cu). In Cs. annulata, the abdominal scales are white colored forming basal bands and the r-m and m-cu veins are joined (Fig. 4A, D), whereas in Cs. subochrea the scales are yellow, almost completely covering the terga, and the r-m and m-cu veins are well separated (Fig. 4B, E). In addition, Cs. annulata presents the C usually completely dark scaled or with isolated pale scales on C, Sc, and R, whereas the Cu is entirely dark scaled and dark spots on wings are distinct. In Cs. subochrea veins C, Sc, and R are with scattered pale scales, some pale scales always present on Cu and dark spots on wings are indistinct. This latter morphological character of the presence of dark and pale scales in the veins is not always easy to distinguish, especially when the individual is damaged. This character in combination with the variability observed in the distance between r-m and m-cu wing veins and the presence of yellow scales in some individuals of Cs. annulata, implied that the best diagnostic character to separate both species is the presence of many scales in the apical half of the terga, a character only observed in Cs. subochrea (Schaffner et al. 2001). Similar evidence of inefficiency of wing characterisation as a diagnostic character to distinguish both Culiseta species has been reported in Balearic Islands (Spain) (preprint; Delgado-Serra et al. 2018).

Identification of females in the subgenus Culicella (Culiseta fumipennis, Cs. litorea, Cs. morsitans, and Culiseta ochroptera) is complex. This includes the different coloration pattern in the middle region of the proboscis, the different coloration pattern of the scales in the abdominal sternites, and the coloration of the tarsomeres of the forelegs (Becker et al. 2010, Medlock and Vaux 2010). In addition, the number of scales in the coxal ventral region was proposed by Encinas-Grandes (1982) to separate Cs. litorea from Cs. fumipennis, and Schaffner et al. (2001) use the coloration of the IV tarsomere of the hind legs to separate Cs. litorea and Cs. morsitans.

In their morphological keys, Becker et al. (2010) distinguish Cs. fumipennis and Cs. litorea from Cs. morsitans and Cs. ochroptera by the proboscis having scattered pale scales in the middle region and the sterna usually with dark scales arranged in an inverted “V-shape.” Cs. fumipennis can be separated further by the forelegs having pale rings, including the apical and basal regions between the tarsomeres III–IV and IV–V, whereas in Cs. litorea the apical regions of tarsomeres III and IV are dark (fig. 6.59a, b in Becker et al. 2010). Conversely, the proboscis is uniformly dark scaled in Cs. morsitans and Cs. ochroptera (although some variation has been observed in these two species), but the sterna is always without a pattern of an inverted “V-shaped” and the pale and dark scales are diffused.

Cs. morsitans can be separated by the terga with narrow basal bands, and the scales of wing veins evenly distribute not forming spots, whereas in Cs. ochroptera the terga does a narrow indistinct basal and apical pale bands (sometimes absent) and the tergum VIII is completely covered with pale scales (fig. 6.58c, d in Becker et al. 2010). Cs. litorea is the only species among the subgenus Culicella, whose apex of the apical lobe longest seta reach the apex of the male gonostyle (Fig. 4F). Furthermore, the complexity in identifying the females of the Culicella subgenus is evident because of variability of morphological characters and the difficulty in observing key diagnostic characters, especially when the specimen is damaged (Cranston et al. 1987, Becker et al. 2010, Medlock and Vaux 2010).

Most of the females analyzed in this study were collected in traps and were in relatively poor condition. In addition, no COI sequences of reliably identified material of Cs. litorea were available in the NCBI or BOLD, and Kuhlisch et al. (2019) state that the COI cannot distinguish between Cs. fumipennis and Cs. morsitans in Germany. Therefore, the examination of the male genitalia is probably the only option to morphologically confirm with certainty the different species of Culicella (Encinas-Grandes 1982, Schaffner et al. 2001).