First record of the little fire ant,Wasmannia auropunctata(Hymenoptera:Formicidae),in Chinese mainland

CHEN Si-qi,ZHAO Yi,LU Yong-yue,RAN Hao,XU Yi-juan

Red Imported Fire Ant Research Center,South China Agricultural University,Guangzhou 510642,P.R.China

Abstract In January 2022,we received ant specimens collected from three field colonies from Shantou City,Guangdong Province,China. They were identified as the little fire ant,Wasmannia auropunctata,through morphological and molecular analyses. Wasmannia auropunctata is listed as one of the 100 most dangerous invasive species by the International Union for Conservation of Nature (IUCN) and has spread from its native range in South America to every continent except Antarctica. DNA analysis of mitochondrial cytochrome c oxidase subunit I (COI) in nine specimens of W.auropunctata found that they had a close genetic relationship with specimens from Argentina. This study represents the first formal record of the establishment of W.auropunctata outdoor in Chinese mainland. However,the invasion stage and occurrence degree of W.auropunctata in China are not clear to date. The implementation of quarantine measures,investigation of the occurrence and distribution,and development of monitoring and control strategies are needed to actively respond to the threat posed by this highly invasive ant.

Keywords:little fire ant,morphological identification,mitochondrial cytochrome c oxidase subunit I

1. lntroduction

The little fire ant,Wasmanniaauropunctata,is listed as one of the 100 ‘world’s worst’ invaders by the International Union for Conservation of Nature (IUCN) (Loweet al.2000). It has spread from its native range in South America to every continent except Antarctica and is distributed in the United States,Argentina,Mexico,Australia,Spain,Israel,and other regions (Wetterer and Porter 2003;Chiffletet al.2018). Many studies have reported important ecological effects ofW.auropunctata,including negative effects on native ant fauna and the diversity of local communities (Le Bretonet al.2003;Walker 2006;Rosselli and Wetterer 2017).Wasmanniaauropunctataattacks hatchling reptiles and birds (Wetterer and White 1999;Jourdanet al.2001;Romanski 2001) and stings people which can cause blindness in indigenous and domesticated animals(Rosselli and Wetterer 2017). This species is considered an agricultural pest due to its behavior of tending of hemipteran insects that transmit plant diseases (De Souzaet al.1998;Wetterer and Porter 2003).

This study is the first formal report of the establishment ofW.auropunctatain the field in Chinese mainland,with specimens initially identified morphologically and later confirmed using mitochondrial cytochrome c oxidase subunit I (COI) DNA analysis.

2.Materials and methods

2.1.lnsect collection

Ant specimens from three colonies were collected by Mr.Liu Zegan from Dongguan University of Technology along the roadsides in Chaonan District (23°15′22′′N,116°26′51′′E,each two colonies apart no more than 10 m),Shantou City,Guangdong Province,in January 2022.Each ant colony contained one queen ant and several worker ants. The ants were immediately soaked in 75%ethanol after collection and sent to the Red Imported Fire Ant Research Center at South China Agricultural University.

2.2.Morphological identification

The workers were first identified to subfamily by morphological features (Bolton 2003);then the ants were identified to the genus level according to the descriptions of Wetterer and Porter (2003),Fisher and Cover (2007)and Longino and Fernández (2007) and finally to the species level using the taxonomic key of Longino and Fernández (2007). Then,we photographed the specimens of workers using a digital microscope (Keyence VHX-5000,Keyence Corporation,Japan).

2.3.DNA extraction,amplification and sequencing

Three workers from each colony were randomly selected for DNA extraction. Genomic DNA was isolated by using a DNeasy?Blood and Tissue Kit (Qiagen,Inc.,Hilden,Germany). The mitochondrially encoded (mt)COI universal primers LCO1490 (5′-GGTCAACAA ATCATAAAGATATTGG-3′) and HCO2198 (5′-TAA ACTTCAGGGTGACCAAAA AATCA-3′) (Folmeret al.1994) were used for PCR amplification.

The TaKaRaTaq? (with Mg2+-free buffer) System(TaKaRa Biomedical Technology Co.,Ltd.,Beijing,China)was used for PCR amplification. The following PCR cycles were used for mtCOI amplification:incubation at 94°C for 5 min;followed by 35 cycles at 94°C for 30 s,52°C for 40 s and 72°C for 50 s;and a final extension at 72°C for 10 min. PCR products were electrophoresed in 1% agarose to verify the integrity of the fragments. Then,these samples were sent to Sangon Biotech (Shanghai City,China) for sequencing.

2.4.Phylogenetic analysis

Sequence data were subjected to BLASTN searches in GenBank (http://www.ncbi.nlm.nih.gov/genbank).Wasmanniarochai(GenBank:EF459824.1) was used as the outgroup for analysis. Distance-based analyses of the COI sequences were performed using MEGA X (Kumaret al.2018). Neighbor-joining trees were constructed based on genetic distances calculated with the Tamura-Nei model. Bootstrap support values were based on 1 000 bootstrap replicates,and only bootstrap values of more than 70% were considered to be supportive(Hillis and Bull 1993). Our nucleic acid sequences were submitted to the GenBank database under accession numbers OM753202-OM753210.

3.Results

3.1.Morphological characteristics

Workers are monomorphic and small to mediumsized,with total length ranging from 1.0 to 1.5 mm.The body is light to golden brown in color and sparsely covered with long,erect setae (Fig.1-A). The head width is significantly larger than the propodeum width. The heads of workers show reticulate sculpture(Fig.1-B). The antenna has 11 segments,with the last two segments greatly enlarged. The antennal scape connects to the head in a distinct groove(scrobe) that extends almost to the posterior border of the head. The clypeus is convex both longitudinally and transversely. The mandibles have five teeth,the maxillary palp is 3-segmented,and the labial palp is 2-segmented (Fig.1-C). The pronotum has angular humeri. The propodeum has reticulate sculpture,is punctate and has long and sharp spines. The petiole has two segments with reticulated puncta. The petiole is “hatchet-like”,with a node that is almost rectangular in profile and higher than the postpetiole. The petiole has a sharp ventral process and is wider than the postpetiole,which also has a ventral process. The gaster is smooth and shiny,with a well-developed stinger (Fig.1).

Fig.1 Lateral (A),dorsal (B) and anterior (C) views of a Wasmannia auropunctata (Roger) worker (voucher codes:GDST22001,sample for the images is deposited in the Insect Collection,Department of Entomology,South China Agricultural University,Guangzhou,China).

Queens are easily distinguished by their darker,reddish-brown coloration and larger size (approximately 4.5-5.0 mm in length compared to 1.0-1.5 mm in workers). Queens have large compound eyes and three ocelli. The mesosoma is well developed,and the bases of the spines on the propodeum are wide. The dorsum of the gaster at the first segment is large and covers almost the whole gaster. The other morphological characteristics are similar to those of workers.

3.2.COl barcoding

The sequenced fragments of the COI genes obtained from nine individuals were truncated to 570 bp for barcoding.The COI fragment was highly AT biased (67.19% AT on average). Our study is the first molecularly confirmed report of highly invasiveW.auropunctatain China. The BLAST results for all sequences from China indicated a match withW.auropunctata. The highest similarity(99.83%) was found with aW.auropunctatasequence from Argentina (KR106300.1;Fig.2).

Fig.2 Neighbor-joining tree for Wasmannia auropunctata created in MEGA X. Wasmannia rochai (GenBank:EF459824.1)was used as an outgroup.

4.Discussion

This study is the first formal report of the establishment ofW.auropunctatain the field in Chinese mainland;W.auropunctatawas initially identified morphologically and confirmed using mitochondrial cytochrome COI DNA analysis.Wasmannia auropunctatais primarily introduced to new regions through the transportation of infested plants,plant material,and soil (Krushelnyckyet al.2005).According to Zhaoet al.(2018),W.auropunctatain fruits imported from Thailand in 2018 were intercepted by the Lianyungang Customs Office,Jiangsu Province,China. The strong similarity of our sequences withW.auropunctatasequences from Argentina suggests that theW.auropunctatacurrently in China could have originated from South American populations. However,analysis of more samples is required to accurately identify the source of the introduction.

Besides its native range (Wetterer and Porter 2003),W.auropunctatahas been introduced to tropical and subtropical areas like Gabon and Cameroon in Africa,Israel in the Middle East (Vonshaket al.2009),North America and South America,Australia,and some islands in the Caribbean and the Pacific Ocean (Holwayet al.2002;Xuet al.2021) and very recently in Taiwan of China (Leeet al.2021). The genetic algorithm for rule set production (GARP) was applied to estimate the potential distribution ofW.auropunctatain China. The results indicated thatW.auropunctatacould occur in most of the southeastern area and limited parts of the northwestern area of China (Chenet al.2007). With the continuous increase in international trade,the risk of introduction and dispersal ofW.auropunctatain China is increasing.

Introduced populations ofW.auropunctataare well documented to reduce the diversity of native arthropods. In New Caledonia,W.auropunctatahave had major impacts on native ants,spiders and lizards(Le Bretonet al.2003). Other studies have found thatW.auropunctatacan have direct effect on tortoises,birds (Loweet al.2000;Wetterer and Porter 2003),domestic dogs and cats (Wetterer and White 1999) and their wild relatives (Walshet al.2004). Once an alien species is detected,it has generally passed the stage of introduction and is in the stage of successful population establishment or even dispersal. This makes it important to create awareness among the public and govenment about the high risks that this species may pose to Chinese biodiversity,economy and livelihood.

Given the ecological importance of this pest,we recommend a rapid eradication of the Shantou populations ofW.auropunctatawhile they might be in the early phase of invasion. Also,a thorough monitoring of potential habitats in port cities in southern China should be established as they appear to be one of the main potential entrance zones for little fire ants. Investigation of its occurrence and distribution,and development of monitoring and control technologies are also needed in the future to actively respond to the threat posed by this highly invasive ant.

5.Conclusion

This is the first report ofW.auropunctatain Chinese mainland based on morphological and molecular analyses.Wasmanniaauropunctataattacks pets,domestic animals,and wildlife,and stings people which can cause blindness. Considering the presence ofW.auropunctatain Chinese mainland,monitoring and rapid eradication programs of this invasive species is required as early as possible.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was funded by the National Key Research and Development Program of China (2021YFC2600404). We are grateful to Mr.Liu Zegan from Dongguan University of Technology,China for his help with sample collection in this study. We are also grateful to Dr.Liu Weixin from South China Agricultural University for her help in photographing the specimens.