First record of the lawn chinch bug Blissus insularis Barber (Hemiptera: Blissidae) in Europe

Since October 2019, chinch bugs have been detected in large sunken dead patches of St. Augustinegrass (Stenotaphrum secundatum) lawns in different locations of Lisbon and Setubal districts, in Portugal. Based on morphological and molecular studies, using mitochondrial Cytochrome C Oxidase subunit I (COI) gene, the collected insects were identified as Blissus insularis (Hemiptera, Heteroptera, Blissidae). This is the first record of this noxious chinch bug species in Europe and in the Palaearctic region.


Introduction
The St. Augustinegrass, Stenotaphrum secundatum (Walter) O. Kuntze (Poaceae: Panicoidae), is a perennial grass that includes fertile diploids and sterile polyploids. Since the beginning of botanical explorations, the common diploid type has been found as a coastal pioneer on both sides of the Atlantic and later one triploid form of this species first appeared in the Cape of Good Hope region (Sauer 1972). St. Augustinegrass was introduced in Asia, Australia, New Zealand, Pacific islands and some areas of USA and Europe, mainly with horticultural purposes, and became a popular turf grass in many of the warmer regions of world, for its climatic adaptation and tolerance to different conditions, from full sun to moderate shade (Sauer 1972;Macfarlane and Shelton 1986;Cherry 2001;Aldous et al. 2014;OGTR 2018). In Portugal, besides being commonly used in public and private lawns, mainly in central and south regions, S. secundatum is occasionally found as a subspontaneous plant in humid or salty places (Franco and Afonso 1998).
When properly planted and maintained, a healthy and vigorous lawn of St. Augustinegrass produces a dense carpet of textured grass blades and has a high capacity to adapt to the soil and local environmental conditions, supporting small populations of insect pests, such as weevils, web worms, army worms, cut worms, or nematodes. However, chinch bugs (Hemiptera, Heteroptera, Blissidae) can cause significant damage to St. Augustinegrass lawns in many regions (Kerr 1966;Sweet 2000). Chinch bugs are sap-sucking species of the genus Blissus Burmeister that feed on phloem tissues above and below the soil surface, causing gradual yellowing and eventual dead patches of turf (Leonard 1966). These insects seem to prefer open sunny areas, especially those with abundant thatch (Vázquez and Buss 2006;Reinert et al. 2011).
Blissus insularis is considered the most damaging pest of St. Augustinegrass (Cherry 2001), being able to build up very large populations, with more than 2000 insects/0.1 m 2 (Reinert and Kerr 1973). Nymphs and adults of B. insularis are phloem feeders on S. secundatum causing wilting, chlorosis, stunting and death of damaged plants. At the beginning of the attacks, damage is limited to small patches of dead grass, and eventually expands, with entire lawns killed as time progresses (Vázquez and Buss 2006;Reinert et al. 2011).
Adult members of the genus Blissus are small and somewhat elongated insects, measuring 2.0 to 4.0 mm long and 1.0 mm wide, with a ratio of length/width between 2.8 and 3.1 (Leonard 1966). The antennae are long and slender, consisting of four segments. Females are slightly longer and larger than males and have an obvious slit on the rounded abdomen, where the ovipositor rests instead an oval plate as in males. The head is usually narrower than the posterior margin of the pronotum. They are fuscous to black in colour with black and white wings, antennae variously infuscate, legs reddish-yellow, often with a dark burnt orange tint. The body is setose with somewhat recurved shorter setae and often less dense long erect setae. Wing dimorphism occurs within the genus, so populations can consist of long-winged forms (macropterous), in which the wings reach almost the end of the abdomen, and shortwinged forms (brachypterous), where the wings extend for less than half the length of the abdomen, or both. They have a long rostrum with four segments and the tip reaches at least the level of mesocoxae (Leonard 1966;Slater 1979;Schuh and Weirauch 2020).
The taxonomic identification of lawn chinch bug is based on the adults' body size, pubescence, general colour patterns, the colour of the posterior and anterior lobe of the pronotum, mouthpart morphology (total length and length of individual segments), complemented, in some cases, with host range and geographic distribution (Leonard 1966(Leonard , 1970Slater and China 1961;Slater and Baranowski 1990;Hoffman 1996;Sweet 2000;Anderson et al. 2006;Larson and Scudder 2018).
In October 2019, the Laboratório de Patologia Vegetal "Veríssimo de Almeida" (LPVVA), School of Agriculture, University of Lisbon, was asked to investigate a new phytosanitary problem in St. Augustinegrass lawns, at Seixal in Setubal district. Samplings in the affected areas ( Fig. 1) showed the damaged lawns were associated with large numbers of chinch bugs that were identified as B. insularis. Here we report for the first time the presence of this chinch bug in Portugal and present the results of a survey carried out in Lisbon and Setubal districts, to assess its distribution in the region.
Adults and nymphs of chinch bugs ( Fig. 2a and Fig.  2b) were collected on damaged lawns of St. Augustinegrass, placing a 4-6 cm × 4-6 cm square of lawn in a bucket full of water and waiting for them to float to the top (Shetlar and Andon 2012). The collected specimens were preserved in ethanol 70% for morphological and molecular studies. Voucher's specimens were deposited in the collection of LPVVA.

Morphological studies
For species identification, four adult females and four adult males (macropterous form), from each of the 10 studied locations (i.e., 80 insects), were studied under stereomicroscope (Leica MZ 12.5), and their images were captured at magnifications from ×20 to ×62.5 with a camera mounted in a system with motorized z-axis drive and inbuilt montaging software (Leica MC 170 HD). For each studied specimen, the general colour pattern and the colour of the posterior and anterior lobe of the pronotum were registered. The body length (from the tip of the clypeus to the posterior-most part of the body), body width (maximum width across humeral angles in dorsal view and maximum width of body in ventral view at right angles to the body length) and rostrum length were measured, using the software Leica Application Suite version 4.12.0. In addition, the rostral segments lengths of six females and six males were measured in wet mounted microscope slides under an optical microscope (Leica DM 2500) equipped with a reticule eyepiece calibrated with a stage micrometer.
The identification of the studied specimens was based on Slater and Baranowski (1990) and Anderson et al. (2006).

Molecular studies
For the amplification of the mitochondrial Cytochrome C Oxidase subunit I gene (COI) total DNA was extracted from legs of two specimens collected at Seixal and two specimens collected at Almada, using the DNeasy Blood and Tissue® kit (QIAGEN, Hilden, Germany). The protocol provided by the manufacturer was followed, with the following modifications: initial sample washed with ultrapure sterilized water, followed by instant freeze in liquid nitrogen to improve cell lysis and addition of 4 μL of RNase A after the incubation period (approx. 1 h at 56°C). Polymerase Chain Reaction (PCR) was conducted using the primers LCO1490 5'-GGTCAACAAATCAT AAAGATATTGG-3′ and HCO2198 5'-TAAACTTC AGGGTGACCAAAAAATCA-3′ (Folmer et al. 1994)‚ which extracted a fragment of 658 bp of the COI region. Each 25 μL PCR reaction tube contained 12.5 μL of DreamTaq™ MasterMix (2x) solution (Thermo Fisher Scientific, Lithuania), 9.5 μL of ultra-pure sterilized water, 1 μL of each specific primer (each with a concentration of 10 μM) and 1 μL of DNA (approx. 5 ng). The PCR cycle program was as follows: initial denaturation at 95°C for two minutes, followed by 40 cycles of denaturation at 95°C for half a minute, annealing at 46°C for a minute, extension at 72°C for one minute and a final extension at 75°C for five minutes. PCR cycles were conducted on a S1000 Bio-Rad Thermal Cycler. Quality of the PCR reactions was verified on an agarose gel (0.5X TBE). DNA sequences were obtained through Sanger sequencing protocol. Amplification of COI fragments was made on an ABI 3730 XL sequencer at Stabvida (http://www. stabvida.com). Each sequence was blasted against the sequences of Blissus species available in GenBank (www.blast.ncbi.nlm.nih.gov) for a species match.

Observed symptoms and damage
At all the sampled lawns, symptoms included stunting (failure of leaf sheaths and internodes to elongate properly), reddish to yellowish feeding marks behind leaf sheaths and a yellowish streaking on the leaves. Sunny areas were often most heavily infested. Severely damaged plants died or were badly stunted, with leaves at the growing point wilted or dead (Fig. 1).

Morphological studies
The collected adults of chinch bugs consisted of both macropterous and brachypterous forms (Fig. 2a). They are grey to black and hirsute, with the body covered with fine silver-grey semi-erect hairs. The pronotum posterior lobe is black, strongly contrasting with the grey colour of the anterior lobe (Fig. 2c). In the macropterous adults, the wings are white, with a distinctive triangularshaped black marking in the middle of the outer edge of each wing (Fig. 2d).
In the macropterous specimens, the adult female and male lengths ranged from 3.03 to 3.78 mm (3.57 ± 0.14 mm) and from 2.84 to 3.50 mm (3.19 ± 0.13 mm), respectively. The abdomen of females was slightly longer and larger than that of males, with the resting ovipositor instead an oval plate as in males (Fig. 2e). Adult females were 0.98 to 1.16 mm (1.05 ± 0.06 mm) wide, whereas adult males were narrower, ranging from 0.80 to 0.99 mm (0.92 ± 0.04 mm). The maximum width across humeral angles in dorsal view ranged between 1.07 and 1.19 mm (1.11 ± 0.4 mm), in females and between 0.95 and 1.01 mm (1.00 ± 0.2 mm), in males. Fig. 1 Damage caused by Blissus insularis on a St. Augustinegrass lawn: left -infested lawn displaying discolored patches, which are usually circular in shape; right -detail of the initial stage of a discolored patch of lawn The rostrum extends at least below the attachment point of the third pair of legs and sometimes the tip reaches the abdomen (Fig. 2f). Overall, rostrum length was higher in adult females, than in males. The rostrum length measured between 1.44 and 1.77 mm (1.62 ± 0.07 mm), in females and between 1.41 and 1.71 mm (1.50 ± 0.06 mm), in males. The mean length of the four individual rostrum segments differed between genders: 409.7 μm and 370.5 μm (segment 1); 525.2 μm and 478.2 μm (segment 2); 351.4 μm and 329.3 μm (segment 3); and 377.5 μm and 349.3 μm (segment 4), for females and males, respectively.

Molecular studies
The 633-bp sequences derived from three of the studied individuals (Almada sample) were 100% identical to each other (GenBank accession MW435183), whereas the sequence of the other one (Seixal sample) had 99.69% similarity to the others, with two singlenucleotide polymorphisms (SNPs) (GenBank accession MW435184). Based on a megablast search of NCBI GenBank nucleotide database, the closest hits using both COI sequences had highest similarity to an uniden-

Taxonomic identification
The morphological characteristics of the studied specimens of the lawn chinch bug collected in Portugal are in accordance with those described for B. insularis (Slater and Baranowski 1990;Anderson et al. 2006). The results of the molecular studies confirmed they belong to the genus Blissus, but did not allow the identification at the species level. In fact, previous analysis of the COI gene of heteropteran species by several authors (Jung et al. 2011;Raupach et al. 2014;Kim and Jung 2018) revealed that the mean maximum intraspecific distance of the compared species was around 1.8%, while the average minimum interspecific distance of congeners was 3.5%. According to Park et al. (2011), these values are congruent with the enormous diversity of this taxon, where there are countless species ordinarily morphologically similar and closely related.
The extensive damage observed in St. Augustinegrass lawns supports our identification since, as mentioned earlier B. insularis is the only Blissus species known as a common pest of St. Augustinegrass.

Concluding remarks
The presence of B. insularis was confirmed in all 10 sampled locations, which indicates that this alien chinch bug has already established and dispersed within an area whose limits are 13 Km North, 26 Km West and 38 Km Southeast from Lisbon. Eight of the sampled locations are up to 26 Km from Lisbon, whereas the other two locations are up to 21 Km from Setubal. Both Lisbon and Setubal have international ports, and Lisbon has an international airport. The importation of contaminated plants was the possible pathway of invasion. This is the most important pathway of alien Heteroptera in Europe (Rabitsch 2008).
According to Rabitsch (2008Rabitsch ( , 2010, at least 16 Heteroptera species are alien to Europe, of which about 63% are of North America origin. No Blissus species is included in that list. To the best of our knowledge, the documented presence of B. insularis in Portugal corresponds to the first report of this species both in Europe and in the Palaearctic region. The severe damages caused by this chinch bug in different locations of Lisbon and Setubal districts demand further studies on its bionomics and the development of effective pest management strategies. Pest management of B. insularis populations has been mostly based on the application of insecticides and use of resistant cultivars of St. Augustinegrass, such as 'Floratam', 'Floralawn', 'FX-10' and 'Captiva'. However, the existence of resistant populations of the chinch bug has been reported (Genovesi et al. 2009;Reinert et al. 2011;Vázquez et al. 2011;Milla-Lewis et al. 2017).