The Asian tiger mosquito Aedes albopictus (Skuse) in Kosovo: First record.

The Asian tiger mosquito, Aedes albopictus, is an invasive mosquito species that is considered a potential vector of about 22 arboviruses, among which dengue, chikungunya and Zika. Here we report the first record of Aedes albopictus in the territory of the Republic of Kosovo. The first finding, in July 2020, was driven by a photo of an adult mosquito published by a citizen in social media. The subsequent field investigation in July 2020 confirmed the presence of adult mosquitoes by human landing catch and collection of eggs in ovitraps at the village Zhur. Monitoring was performed for 7 weeks with ovitraps and BG-Sentinel adult traps at 36 sampling stations from 7 localities, in the Southern part of Kosovo, on the border with Albania. Fifty-two out of 81 ovitrap samples resulted positive for the presence of Ae. albopictus. A total of 2,711 eggs are collected in 22 out 36 stations and a total of 78 adults from 4 out 4 stations with BG Sentinel trap and 15 adults with handled electric aspirator. Our finding shows that the tiger mosquito is expanding its geographical range in the Balkans, southeastern Europe.

Introduction

Aedes (Stegomyia) albopictus (Skuse, 1894) (Diptera: Culicidae), commonly called the ’Asian tiger mosquito’, is widespread throughout the tropical and subtropical regions of the world. During the last 30 years, it has invaded many countries worldwide, including in the Mediterranean Basin. The species is currently considered one of the top 100 invasive species globally and the most widespread invasive mosquito species in Europe [1, 2]. It represents a severe threat to public health due to its aggressive daytime human-biting behavior and its vectorial competence for numerous arboviruses of the family Flaviviridae (e.g., dengue, West Nile, and Japanese encephalitis viruses), Bunyaviridae (e.g., Rift Valley fever, Potosi, Cache Valley, and La Crosse viruses), Togaviridae (e.g., chikungunya and Ross River viruses) [3-7]. After Ae. aegypti, Ae. albopictus is the secondary vector of dengue and dengue hemorrhagic fever [8]. In Europe, the species was incriminated as the vector in outbreak of, among others, chikungunya in 2007 northeastern Italy [9], dengue in 2010 in Croatia [10], dengue and Zika cases between 2010 and 2020 in France [11-13].

The first report of the tiger mosquito in the European continent dates back to 1979 in Albania [14]. It is thought to have been imported in shipments and containers from China in the mid-1970s. Later it has been detected in Italy in 1990 and has spread throughout the European continent via various routes [1]. In the surroundings of Kosovo, this species was recorded in Montenegro [15], in North Macedonia [16], and Serbia [17]. In Serbia, Ae. albopictus was intercepted in two districts in the western and southwestern part of the country. It has been present for the past nine years on the Croatian border (Batrovci, northwest of Serbia) [18], and on the Montenegro border since 2014 [19].

Distribution models predict that Ae. albopictus will continue to expand, depending on transport, environmental, and climatic changes [20-22].

This worldwide expansion is mostly based on transport and dissemination of dormant egg via the international trade of used tires [23] and shipments of the Asian plant ‘‘lucky bamboo” (Dracaena spp.) [24, 25] and by public and private ground transport from heavily infested areas [22]. In 2017, a research for tiger mosquito in Kosovo was conducted at the borders with Macedonia and Albania within the VectorNet project framework. It resulted negative, although Kosovo showed favorable conditions for the development of this mosquito species [26].

The primary objective of our study was, following a citizen report, to confirm the presence of the tiger mosquito in the territory of Kosovo and to determine its distribution.

Materials and methods

Study area

The present study was conducted in the municipality of Prizren (42.2166 N, 20.7333 E) and in the city of Suhareka (42.2248 N, 20.2248 E) in July, August and September 2020. The Municipality of Prizren, occupies the southern position in the Dukagjini Plain and southwestern Kosovo. The average altitude is about 450 m above sea level and includes nearly 640 km2 of the Kosovo’s entire surface. Sharri mountain, Prizren plain and downstream area of the Drini I Bardhë define the relief. Sharri mountain is also an essential geographical element since it constitutes a watershed between the Adriatic Sea and the Aegean Sea basin. The climate of this part of Kosovo is classified as Mediterranean. The city of Suhareka is located in the southern part of Kosovo. The average altitude is about 455 m. It is characterized by a rich hydrography.

Collection and identification

We used ovitraps which are commonly used for detecting the females’ presence via egg laying [27], and entomological aspirator (GeniccoSrl, Italy, Model: JF0825S1H—R) and BG-Sentinel™ traps (Biogents, Germany) to collect the adult mosquitoes. The ovitraps (500 ml black plastic cups) were filled with tap water and equipped with a masonite strip (12.5 × 2.5 cm) for egg deposition. The plastic cups were modified by two holes, punching 3 cm from the top of the cup to prevent water overfilling. A total of 36 ovitraps were randomly distributed in 7 localities [Vërmicë (5), Zhur (11), Vlashnje (5), Prizren (8), Atmaxhë (3), Landovicë (3) and Suharekë (1)]. The distance between the traps was 100 m at minimum. At four localities (Vërmicë, Zhur st.1, Zhur st.2, Prizren) we also used BG-Sentinel traps baited with BG-Lure and CO2. The ovitraps were placed on the ground, in shaded and accessible places, under vegetation, with free space above at least 1 m (Fig 1A–1D).

Fig 1

Examples of traps operating at several mosquito collection sites.

Ovitraps in: a Vlashnje (Tyrecentres); b Zhur st.1 (Privat residence garden); c Prizren (Privat residence garden); d Vërmicë (Restaurant veranda); e Odour-baited adult traps (BG-Sentinel) Vermice (Restaurant garden); f Zhur st.1 (Resident garden); g Prizren (Resident garden). Catching with aspirator: h Vlashnje (Tyrecentres). Adults resting in: i Prizren (on the human body); j Zhur (Plastic bottle); k Vlashnje (Inside the tire, resting on the surface water); l Zhur st.1 (first specimen of Aedes albopictus caught while landing on human body).

The ovitraps were left at the same place during a sampling period of 10 successive days. After 10 days, the masonite strips were collected from the traps and transferred to the laboratory at the university of Prishtina for identification based on their color, size, shape and surface sculpting [28]. The adult mosquitoes were identified using the MosKeyTool identification key [29]. Another technique used in this research was aspiration from human bait. This technique was used in three localities (Zhur st.1, Vlashnje, Prizren, (Table 1) for 30 minutes (Fig 1H, 1I and 1L).

Table 1

Results of Aedes albopictus adult trapping with BG-Sentinel traps (BG) or handheld electric aspirators (A) in the Prizreni municipality (July -September 2020).

Locality	Latitude (N)	Longitude (E)	Method of capture	Sampling area	Sampling period#	No. of adults	No. of adults
						Ae. albopictus	Cx.pipiens s.l
Vërmicë	42.166918	20.572473	BG	Restaurant garden with vegetation	25-07-20	0	7
					09-08-20	15	3
					25-08-20	12	3
Zhur st.1	42.16604	20.61539	BG	Private residence garden	25-07-20	27	3
					09-08-20	10	0
					25-08-20	5	0
Zhur st.2	42.161245	20.623351	BG	Private residence garden	25-07-20	0	2
					09-08-20	4	5
					25-08-20	0	0
Prizren	42.223997	20.734394	BG	Private residence garden	25-07-20	0	2
					09-08-20	2	5
					25-08-20	3	7
Zhur st.1	42.168164	20.615606	A	House yard	01-08-20	1	0
					10-08-20	3	0
Vlashnje	42.198731	20.667758	A	Tire storage	03-09-20	9	0
Prizren	42.223997	20.734394	A	House with vegetable garden	30-08-20	1	0
					08-09-20	1	0

#Entomological survey period: I: 25–27 July 2020; II: 08–10 August 2020; III: 25–27 August 2020.

One BG-Sentinel trap was run for two consecutive nights every two weeks and for three periods (Table 1). The traps were set in the private houses’ backyard (Fig 1E–1G). In total we did perform 12 BG-Sentinel trap-nights.

Results

The presence of Ae. albopictus was registered in Kosovo for the first time at the end of July 2020 in Zhur village. The annoyance and bites that the mosquito caused to citizens during this period encouraged citizens to post a mosquito photo on a social media. This information suggested the tiger mosquito to be present in this area and to perform a field survey. Our first catch, a male mosquito (Fig 2), was caught by hand on 23.07.2020 in a private residence garden at Zhur st.1 (Table 1). It was identified in the laboratory of the Institute of Public Health, Tirana, Albania, as belonging to Ae. albopictus.

Fig 2

First specimen of Aedes albopictus caught in Zhur st.1, date 23.07.2020 (photo: N. Muja-Bajraktari).

In total, 52 ovitrap samples revealed positive, yielding 2,711 eggs collected at 36 sampling stations from 7 localities (Fig 3).

Fig 3

Distribution map of the ovitraps in Prizreni region showing positive and negative site for the presence of Aedes albopictus.

The general administrative maps were extracted from the Natural Earth (https://www.naturalearthdata.com/) and then modified subsequently according to the information presented in the map, using Q-GIS version 3.18.

Using ovitraps, we collected eggs of Ae. albopictus but also of the native species Aedes (Dahliana) geniculatus (Olivier, 1791) (Table 2) at the Prizreni municipality. Eggs of Ae. albopictus were found at 22 out of the 36 sampling stations, while 14 remained negative. We did collect 440 eggs of Ae. albopictus at Vërmicë, 1,187 at Zhur, 139 at Vlashnje and 119 at Atmaxha, respectively. In the city of Prizren we collected 786 eggs from three stations, while 40 eggs were counted from ovitraps in 3 stations at a Truck Terminal, on the periphery of the city of Prizren. We didn’t found any egg in the Landovica and Suhareka city (Table 2).

Table 2

Location of ovitraps in the Prizreni municipality and numbers of eggs collected for Aedes albopictus and Aedes geniculatus.

Localities	ID	Sampling period#	Latitude (N)	Longitude (E)	Sampling area	No. of eggs
						Ae. albopictus	Ae. geniculatus
Vërmicë	01/1	I, II, III	42.167278	20.577672	Near the road	68	63
	01/2	I, II, III	42.168801	20.582681	Near the road	54	0
	01/3	I, II, III	42.166918	20.572473	Restaurant garden	202	0
	01/4	I, II, III	42.164574	20.567957	Restaurant	112	0
	01/5	I, II, III	42.163792	20.563543	House veranda	4	0
Total						440	63
Zhur	02/1	I, II, III	42.168164	20.615606	Private residence garden	346	0
	02/2	I, II, III	42.166543	20.607746	Private residence garden with pets	196	0
	02/3	I, II, III	42.161062	20.622807	Private residence garden	168	48
	02/4	I, II, III	42.15736	20.618477	Private residence garden	62	0
	03/1	I, II, III	42.165116	20.623798	Private residence garden	175	0
	03/2	I, II, III	42.161867	20.611347	Private residence garden	240	0
	03/3	I, II, III	42.223801	20.734074	Private residence garden	0	0
	03/4	I, II, III	42.15956	20.629809	Private residence garden	0	0
	04/1	IV	42.163807	20.632446	Private residence garden with chicken	0	0
	04/2	IV	42.166677	20.627916	Private residence garden	0	0
	04/3	IV	42.15726	20.624888	Near forest	0	0
Total						1,187	48
Vlashnje	05/1	II, III	42.200968	20.660929	At the gas station near the road	76	0
	05/2	II, III	42.204696	20.659614	Inside the tires	30	0
	05/3	II, III	42.201094	20.664804	Inside the tires	33	0
	05/4	II, III	42.203438	20.668572	Tire center garden	0	0
	05/5	II, III	42.200968	20.660929	Tire center garden	0	0
Total						139	0
Prizren	06/1	I, II, III, IV	42.223288	20.741278	Private residence garden	438	4
	06/2	I, II, III	42.228297	20.739493	Private residence garden	176	0
	06/3	I, II, III	42.226967	20.729043	Private residence garden	172	0
	06/4	I, II, III	42.223801	20.734074	Private residence garden	0	0
Total						786	4
Truck terminal	07/1	IV	42.249954	20.729769	Truck terminal garden	3	0
	07/2	IV	42.256972	20.733839	Restaurant garden	16	0
	07/3	IV	42.254285	20.72666	Near the road	0	0
	07/4	IV	42.250268	20.721548	Truck terminal garden	21	0
Total						40	0
Atmaxhë Total	08/1	III	42.245329	20.699713	At the gas station near the road	73	0
	08/2	III	42.24801	20.696709	Near the car wash	46	0
	08/3	III	42.243715	20.702604	Hotel garden	0	0
						119	0
Landovicë	09/1	IV	42.259853	20.688001	Supermarket forecourt	0	0
	09/2	IV	42.264082	20.6842	Near the road	0	0
	09/3	IV	42.255862	20.684525	Private residence garden	0	0
Suharekë	10/1	I, II, III, IV	42.363973	20.832803	Bus station	0	0
Overall total						2,711	115

#Entomological survey period: I: 23–02 July 2020; II: 02–12 August 2020; III: 12–22 August 2020; IV: 02–12 September 2020.

Eggs of Ae. geniculatus were found only at 3 sites from 3 locations, i.e. Vërmicë, Zhur and Prizren (Table 2).

Four adult females were collected with an aspirator at a house yard, nine adult mosquitoes at a tire center with numerous tires holding rainwater, (Fig 1h and 1K) and two males at a house vegetable garden. Using BG-Sentinel traps, we collected 78 adult mosquitoes (38 females and 39 males) at four sampling stations in gardens of residential houses or restaurant within three entomological survey periods (Table 1).

Forty-eight eggs were successfully hatched and were reared to adults (18 males / 30 females). All adult mosquitoes were morphologically identified and classified as Ae. albopictus. During the research period, we also identified 115 eggs of Ae. geniculatus caught with ovitraps (Table 2) and 34 adult mosquitoes of Culex pipiens s.l. Linnaeus, 1758 caught with BG-Sentinel trap. (Table 1).

Discussion

The Asian tiger mosquito Ae. albopictus, originating from Southeast Asia, has undergone a significant expansion of its range in the last few decades [30].

In Europe, the tiger mosquito was first reported in Albania in 1979 [14], than ten years later in Italy [31], France [32, 33], Spain [34], Belgium [35], Switzerland [36], Greece [37], Montenegro [17, 38], Croatia [15], Bosnia and Herzegovina [15], Slovenia [15, 39] and North Macedonia [16]. The movement of cars has helped a lot in distributing Ae. albopictus species [40]. The first identification of the tiger mosquito was made near the border with Albania. It is thought that the way of its introduction was done through land routes strictly through the movement of vehicles.

Our study reports an established population of the Asian tiger mosquito in the municipality of Prizren in the southern part of Kosovo.

The ovitraps were selected as a research method due to their high sensitivity to low mosquito density, low price and practical use in the field [41]. A female tiger mosquito can lay eggs in several ovitraps placed in different areas; however, it depends on the sites’ attractiveness [42]. Ovitraps can help control the mosquito population by eliminating the eggs, which results in a lower number of mosquitoes [16]. The first Ae. albopictus specimen is registered in Zhur, a village close to the border with Albania. This specimen recorded in a garden with many artificial breeding places (for container-breeding species). The ability of the tiger mosquito to use both natural and artificial containers for larval habitats facilitates the widespread occupation of urban and peri urban environments [30], ensuring a close connection between the species and the human population and increasing the risk of vector-borne disease pathogen transmission in these areas [43].

The other sampling station, Vërmicë, is the closest to Albania’s border, which argues the significant presence of eggs in the ovitraps and the large number of adults caught with BG-Sentinel traps. The border zones are considered highly vulnerable for introduction of potential invasive species. The number of cars and other vehicles coming from Albania stop for a considerable time at the border crossings, respectively in Vërmicë, so introducing the tiger mosquito through this road is indisputable. After that, from the border with Albania continues the highway, Morinë-Prizren-Prishtinë, therefore it is expected that mosquitoes will spread in other parts of Kosovo, precisely through the land route. Also, the third sampling station in Vlashnje is characterized by the significant presence of used tires, which served as breeding sites. At the monitoring station inside the city of Prizren, the number of eggs in ovitraps was high, suggesting presence of a high number of adults. The recording of Ae. albopictus in this part of Kosovo is an important finding, demarcating new boundaries of the distribution range of the species in the Balkans and suggesting an increasing mosquito-borne disease threat to public health in that region. Based on our results, Ae. albopictus may be, by 2020, absent from the Landovica locality, in the periphery of Prizren, and from Suhareka, located about 41 km away from the Albanian border.

As a potential invasive species, it is expected that Ae. albopictus will expand its distribution to other parts of the country. This already happened with other invasive insect species recorded in Kosovo like the western conifer seed bug Leptoglossus occidentalis. This insect was reported from two localities in the east of the country, in the area of Batllava Lake, as well as in two localities from the foothills of Bjeshkët e Nemuna Mountains, in the western part of Kosovo [44].

Conclusions

Since the presence of the tiger mosquito in the Republic of Kosovo is confirmed, it is expected that the Health authorities will develop a monitoring system for both mosquito and pathogen surveillance to survey and prevent the risk of pathogen transmission. Community involvement has been very successful in other countries, therefore we suggest to further encourage community involvement (citizen scientists) for an early detection of this invasive mosquito species in other parts of the country.

Results of the investigation for the presence of Ae. albopictus eggs at 36 stations in Kosovo and Ae. albopictus adult trapping with BG Sentinel traps (BG) or handheld electric aspirator (A).

(XLSX)

Click here for additional data file.

29 Nov 2021

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Carina Zittra, Ph.D., Mag.rer.nat.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

3. We note that Figure 3 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 3 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

4. We note that Figure 1 includes an image of a [patient / participant / in the study].

As per the PLOS ONE policy (http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research) on papers that include identifying, or potentially identifying, information, the individual(s) or parent(s)/guardian(s) must be informed of the terms of the PLOS open-access (CC-BY) license and provide specific permission for publication of these details under the terms of this license. Please download the Consent Form for Publication in a PLOS Journal (http://journals.plos.org/plosone/s/file?id=8ce6/plos-consent-form-english.pdf). The signed consent form should not be submitted with the manuscript, but should be securely filed in the individual's case notes. Please amend the methods section and ethics statement of the manuscript to explicitly state that the patient/participant has provided consent for publication: “The individual in this manuscript has given written informed consent (as outlined in PLOS consent form) to publish these case details”.

If you are unable to obtain consent from the subject of the photograph, you will need to remove the figure and any other textual identifying information or case descriptions for this individual.

5. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors describe the first records of tiger mosquitoes in Kosovo. The manuscript is of high relevance in the field and provides novel information.

Major comment: English language editing is needed.

Minor comments:

entire document – e.g. Line 31: Change Aedes to Ae. when needed (not at first mentioning and at beginning of sentences)

The definition “invasive” should be used with care. It has not proven to outcompete native mosquitoes in Kosovo. It is recommended to change to “potential invasive”

Line 64: Mosquitoes transmit pathogens and not diseases.

Line 82: Dracaena in italics

Line 109: The link to BG is not necessary. It is better to mention BG-sentinel generation 2. Moreover it is not mentioned if CO2 and lure were used. Info is give in line 139 – recommended to move to line 109

Line 182: s.l. not in italics

Line 180-190: Many countries are missing – if all European countries where Ae. albopictus was reported are planned to be listed

Mosquito alert is mentioned but no data shown. Maybe discuss that this app might increase the number of reports (the reviewer has seen tiger mosquitoes from the Kosovo when specifying for Mosquito Alert)

Reviewer #2: The manuscript is well written and provides interesting findings from Kosovo. There are some issues however to be addressed and most of them are commented directly in the manuscript pdf document. The abstract is somehow too long, and it would be nice to be shortened. In Introduction or somewhere in the Discussion section it would be advisable to give an overview of other invasive insects known from Kosovo, so that the paper is put also in the context of broader invasion challenge. English must be improved as well, although most of the linguistic corrections are done inside the document. I suggest to publish the paper after these revisions.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Halil Ibrahimi

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.