Mosquitoes were collected as larvae and individually reared to adults. Larvae of adults identified as Aedes nigrinus were collected on 22 May 2016 at Beaulieu Airfield (50°48.53'N; 1°29.79'W and 50°48.11'N; 1°30.83'W), New Forest, Hampshire, England. Larvae of Ae. sticticus were collected on 10 May 2011 in Hurcott Wood (32°23.92'N; 2°12.73'W), Kidderminster, Worcestershire, England. The larval and pupal exuviae of Ae. nigrinus were lost; those of Ae. sticticus were mounted in Euparal on microscope slides. Adults were mounted on points on insect pins. Dissected male genitalia of both species were cleared in 5% NaOH for 2 h at 50°C and slide-mounted in Euparal. The pinned adults were examined under simulated natural light with an Olympus SZ6045 stereomicroscope. The dissected genitalia were studied with an Olympus BX50 compound microscope fitted with differential interference contrast optics. Digital images of wings and genitalia were taken with a Canon 550D digital camera mounted on a Leica M125 stereomicroscope and a Zeiss Axioskop compound microscope, respectively; Helicon Focus version 3.03 software (Helicon Soft Ltd, Kharkov, Ukraine) was used to obtain extended-focus images. The anatomical terminology of Harbach and Knight (1980, 1982), revised and updated in the Anatomical Glossary of the Mosquito Taxonomic Inventory (http://mosquito-taxonomic-inventory.info/node/11027), is used in the descriptions and illustrations.

Abbreviations for morphological structures indicated in figures:

BDL basal dorsomesal lobe

C costa

R₁ radius-one

Re remigium

Sc subcosta

1A anal vein

DNA was extracted from two legs from each of five adults of Ae. nigrinus using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) in accordance to the manufacturer’s instructions. Amplification of the mitochondrial cytochrome oxidase subunit I (COI) gene and the nuclear internal transcriber spacer 2 (ITS2) region of ribosomal DNA was carried out using the following primers: 5’- GGATTTGGAAATTGATTAGTTCCTT-3’ (COIF) and 5’- AAAAATTTTAATTCCAGTTGGAACAGC-3’ (COIR) (Chan et al. 2014), 5’- TGTGAACTGCAGGACACATG-3’ (ITS2F) and 5’- ATGCTTAAATTTAGGGGGTA-3’ (ITS2R) (Walton et al. 2007). PCR was undertaken using Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA) and products were purified using Isolate II PCR and Gel spin columns (Bioline Reagents Limited, London, UK). Subsequent sequencing reactions were undertaken using the BigDye™ Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems Inc., Foster City, CA, USA) and sequenced on a 3500 Series Genetic Analyzer. Analysis of the sequence data was carried out using MEGA7 (Kumar et al. 2016). The exact location and size of the ITS2 region was determined by annotation of the 5.8S/28S flanking regions using the ITS2 database (http://its2.bioapps.biozentrum.uni-wuerzburg.de/).

It is unfortunate that the larval and pupal exuviae of mosquitoes reared from larvae collected in the New Forest were lost as these would have aided the identification of Ae. nigrinus. Further field work will be conducted to obtain the immature stages. However, a number of morphological differences are robust enough to distinguish the two species. To aid future identification, the wings and the male genitalia of Ae. nigrinus and Ae. sticticus are illustrated for comparison in Figures 1 and 2, respectively. The principal features that distinguish the two species are as follows.

Figure 1. 

Proximal half of the right wing of female mosquitoes. A Ae. nigrinus, showing the presence of pale scaling on the costa, subcosta, remigium and anal vein B Ae. sticticus, showing the absence of pale scaling.

Aedes nigrinus. A dark mosquito, integument and dark scaling black, well contrasted with pale scaling. Female: first antennal flagellomere and dorsal surface of pedicel black; wing with pale (white) scaling (Fig. 1A) at base of costa (C), length of subcosta (Sc), on remigium (Re) (not evident in figure), base of radius-one (R₁) (not evident in figure) and base of anal vein (1A); abdominal terga with basal pale bands slightly constricted medially, sometimes reduced to lateral patches on tergum VII. Male genitalia (Fig. 2A): basal dorsomesal lobe of gonocoxite more or less globoid in dorsal (tergal) view. Fourth-instar larva (see Natvig 1948, Becker et al. 2010): setae 5,6-C usually single, rarely double; comb usually with 12–16 scales, rarely with more than 20.

Aedes sticticus. A slightly paler mosquito, integument and dark scaling dark brown to brownish black, less well contrasted with pale scaling. Female: first antennal flagellomere and dorsal surface of pedicel yellowish brown; wing entirely dark-scaled (Fig. 1B); abdominal terga mostly without complete basal pale bands, bands on terga II–IV narrow if present, more distal terga with triangular basolateral pale patches. Male genitalia (Fig. 2B): basal dorsomesal lobe more or less crescentic in dorsal (tergal) view, distomesal surface slightly concave. Fourth-instar larva (see Natvig 1948, Becker et al. 2010): setae 5,6-C usually with 2–4 branches, seta 6-C occasionally single; comb with 19–27 scales.

Figure 2. 

Doral aspects (pre-rotation sense) of the male genitalia of Ae. nigrinus (A) and Ae. sticticus (B), which are distinguished by the shape of the basal dorsomesal lobe (BDL).

The female, male and fourth-instar larval stages of Ae. nigrinus and Ae. sticticus have been described, although not completely (Natvig 1948, Becker et al. 2003, 2010), but no attention has been given to the pupal stage of either species. The pupa of Ae. nigrinus remains unknown, but it is possible to distinguish the pupa of Ae. sticticus from other species included the key of Cranston et al. (1987) with the following modification of couplet 20 (measurements corrected and wording changed to reflect current usage of morphological terminology).

Aedes nigrinus and Ae. sticticus were both originally described from localities in Germany (Knight and Stone 1977). As far as known, the distribution of Ae. nigrinus is limited to an area that extends from southern England (new record reported herein) and France to eastern Russia and from southern Scandinavia and Finland southward to Germany whereas Ae. sticticus is broadly distributed in northern areas of the Holarctic Region (Knight and Stone 1977, Becker et al. 2010). According to Cranston et al. (1987), Ae. sticticus has a “patchy distribution” in Europe and “is rare in Britain”. The few specimens present in the NHM collection lack basal pale bands with only lateral pale spots present. Such specimens, like most reared from larvae collected in Hurcott Wood, could be misidentified as Ae. geniculatus (Olivier, 1791) in the key of Cranston et al. (1987).

The occurrence of Ae. sticticus in Britain was first recorded by Marshall (1938), who listed the New Forest among other localities where the species had been collected. The New Forest record was later accepted without question by Cranston et al. (1987) and Snow (1990) and is the only record of the species in southern England (Snow et al. 1998). Since we now know that the New Forest record of Marshall refers to Ae. nigrinus (see above), the New Forest should not be listed as an occurrence record for Ae. sticticus until this species is definitely known to occur there.

The New Forest became a royal forest more than 950 years ago and is the largest remaining tract of unenclosed pasture land, heathland and forest in England. The land is dominated by gravel, sand and clay that was deposited during the Palaeogene Period of the Cenozoic Era (23.03–65.5 Mya). Many sites near the Beaulieu airfield where Ae. nigrinus was collected contain extensive areas of water-logged, marshy bogs and mires where the clay creates an impervious layer of saturated ground. The airfield was established during World War I, closed in 1919, re-opened again in 1942 and operated as an airfield for a further 15 years. Since 1959, the area has returned to an open heathland with open mireland habitat fringing the main airfield site.

Aedes sticticus is primarily associated with floodplains of rivers in forested areas (Cranston et al. 1987, Wood et al. 1979, Becker et al. 2010). In parts of Sweden, it contributes to a significant biting issue and is the subject of an extensive aerial mosquito control programme (Schäfer and Lundström 2009). In other parts of its range in central Europe, it also contributes to significant biting, along with Aedes vexans (Meigen, 1830), in floodwater areas along the Dyje River on the border of the Czech Republic and Austria, and is implicated in the transmission of Tahyna virus (Hubálek et al. 2010, Berec et al. 2014). Larvae of this species were found in a shallow floodwater pool in Hurcott Wood in early May and a few adults were collected in June in forest at Woodwalton Fen, Cambridgeshire. In contrast, Ae. nigrinus is mainly associated with floodwaters in more open terrain, such as meadows (Becker et al. 2010). Larvae of this species were found at the margins of small ponds in an open area of heath within the New Forest National Park in May, and a single adult male was collected in the same area in September.