Migration of invasive Spodoptera frugiperda (Lepidoptera:Noctuidae)across the Bohai Sea in northern China

JlA Hui-ru, GUO Jiang-long , WU Qiu-lin HU Chao-xing, Ll Xiao-kang, ZHOU Xian-yong, WU Kong-ming

1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

2 College of Plant Protection, Shenyang Agricultural University, Shenyang 110866, P.R.China

3 Guizhou Provincial Key Laboratory for Agricultural Pest Management of the Mountainous Region, Institute of Entomology,Guizhou University, Guiyang 550025, P.R.China

4 College of Plant Protection, Southwest University, Chongqing 400716, P.R.China

Abstract The fall armyworm (FAW), Spodoptera frugiperda (J.E.Smith), is an important agricultural pest with a strong migratory ability.While the species is native to the Americas, it has recently invaded China, ravaging crops in many provinces(autonomous regions, municipalities).Its long-distance migration, which is critical to understand for pest management programs, has been well studied in its native region.In other regions however, the moth’s migration patterns have not yet been characterized.Here, the migratory behavior of FAW populations in China was studied on an isolated island located in the center of the Bohai Strait from spring to autumn 2019, the year in which FAW first reached this region, by using searchlight trapping, stable carbon isotopes and trajectory simulation.The main results were summarized as follows:(i) The number of FAW moths caught by searchlight trapping provided direct evidence that the species migrated across the Bohai Sea.(ii) Species identification was confirmed by both morphology and molecular methods, and only the “corn-strain” that preferentially infests maize and sorghum was found in the collections.(iii) Stable carbon isotope measurements showed that up to 95.07% of captured moths displayed the C4 isotope signature, thus providing additional evidence that this species is a migrant as there are no major C4 plants at the trapping site.(iv) Backward trajectory analysis indicated that the pest now threatens to expand its range into the agriculturally important region of Northeast China, and thus authorities in this region should be vigilant to the threat posed by this species.Together, these findings add to our knowledge concerning the occurrence of FAW in northern China, and will help us to develop sustainable and effective monitoring, forecasting, and pest management strategies.

Keywords:Spodoptera frugiperda, overseas migration, searchlight trapping, host strains, stable carbon isotopes, migration trajectory

1.lntroduction

The fall armyworm (FAW),Spodopterafrugiperda(J.E.Smith), is one of the most important noctuid moth pests of cultivated plants in the Western Hemisphere.The moth is noted for its high mobility, broad host range (＞353 host plant species recorded), and strong reproductive capability(Mitchellet al.1991; Pogue 2002; CABI 2016; Westbrooket al.2016).Additionally, the existence of two morphologically indistinguishable but behaviorally different subpopulations or strains, the “corn-strain” and the “rice-strain”, further complicates control efforts (Adamczyket al.1997; Ingberet al.2018).The two strains were initially recognized by differences in the choice of host plant; specifically, the former is preferentially found on maize, sorghum, and cotton whereas the latter predominates on rice and various pasture grasses (Pashley 1986; Pashley and Meagher 1987).

In early 2016, severe outbreaks of FAW were discovered in West Africa, the first time the pest was detected outside of the Americas (Goergenet al.2016; Cocket al.2017).Since then, the moth has spread throughout the continent and has subsequently been found in India and much of southeastern Asia (Stokstad 2017; IPPC 2018, 2019; Nagoshiet al.2018;Sharanabasappaet al.2018).On December 11, 2018,FAW was confirmed to have entered ChinaviaMyanmar,and by October 2019, 26 provinces (autonomous regions,municipalities) had reported it (Jianget al.2019; Sunet al.2021).Such rapid dispersal or invasion poses a significant threat to global food security.For example, a recent report estimated that the damage from this species to African maize production was approximately 4.1-17.7 million tons with a value of USD$1.09-4.66 billion (Stokstad 2017).Since the ability of adult FAW moths to undergo long-distance migration is considered to be the major factor in its rapid wide-scale spread (Earlyet al.2018), investigating the migratory behavior of the pest is essential for developing strategies for managing it in the newly invaded areas.

Long-distance migration, a seasonal movement between different regions where conditions are alternately favorable or unfavorable, is a behavioral adaptation used by numerous insect species (Dingle and Drake 2007; Chapmanet al.2011).Movements of the FAW have been well studied in its native region of North America, where recurring annual seasonal migration occurs over thousands of kilometers(Pair and Spark 1986; Mitchellet al.1991; Nagoshiet al.2012).However, terrain, climate, agricultural practices, and wind patterns could significantly affect migration patterns.In the newly invaded regions, these factors will differ substantially from those in North America.As a result, FAW migratory behavior in other regions, including China, is less well understood, and this presents a considerable limitation for the development of forecasting systems and regional and area-wide controlling strategies for this newly-invaded pest.

Since FAW populations have arrived in the Huang-Huai-Hai Summer Maize Region (mainly the provinces of Henan,Shandong and Hebei), there is a high probability that the pest will further disperse, putting the Northeast Spring Corn Region (Liaoning, Jilin, Heilongjiang and eastern Inner Mongolia) at risk (Wuet al.2019a; Liet al.2020).As these areas are major maize-producing areas in China and are potentially suitable for summer-breeding populations (Earlyet al.2018; Guoet al.2018), an invasion could do significant damage to Chinese agricultural yield and food security.A good understanding of the pattern of this species’ population movements in these regions is therefore urgently needed.

Bohai Strait lies across the major pathway for the seasonal northward and return migration of many insect species in northern China (Fenget al.2003).In an effort to characterize the movement ecology of the FAW in northern China, with the goal of improving regional forecasting and management of this pest, the present study has focused on possible migration of the species across the Bohai Strait.Initially,searchlight trapping on a small isolated island in the center of the sea was used to monitor the high-altitude migration of FAW moths from April to October 2019.Subsequently,for the trapped moths identified as FAW by morphological characteristics, a mitochondrial (CytochromeoxidaseI,COI) and a nuclear (Triosephosphateisomerasegene,Tpi) marker were applied to further confirm, respectively,species identity and host-strain type.Additionally, carbon isotope analysis of wings was carried out to characterize the habitats in which these moths developed.Lastly, likely source regions of the sampled individuals were estimated by backward trajectory analysis.

2.Materials and methods

2.1.Light-trapping and field observation

Searchlight trap monitoring for FAW was conducted every night from April to October 2019 at a field station of the Chinese Academy of Agricultural Sciences on a 2.5-km2island named Beihuang (BH) , located in Changdao County,Shandong Province, China (38°24´N, 120°55´E).The small island is situated in the center of the Bohai Strait and thus lies on the major pathway for the seasonal northward and return migration of many insect species in northern China(Fenget al.2003).The island is approximately 40 and 60 km from the nearest (mainland) agricultural regions to the north and to the south, respectively.

A vertical-pointing searchlight trap (model DK.Z.J1000B/t,Shanghai Yaming Lighting Co., Ltd., Shanghai, China)equipped with a 1 000-W metal halide-lamp (model JLZ1000BT; Shanghai Yaming Lighting Co., Ltd., Shanghai,China), for sampling high-altitude migrants of various insect species (up to ～500 m above ground level), was placed on a platform ～8 m above sea level (Fenget al.2009; Feng and Wu 2010).

Since the searchlight trapping commenced in 2003,the lamp has been turned on at sunset (from 17:15 on 31 October to 19:30 on 22 June) and turned off at sunrise (from 4:45 on 22 June to 6:40 on 31 October) every day (except for times of power outage or heavy rain).Trapped insects were collected with a nylon net bag (60-mesh) beneath the trap,and the bag was changed manually every 2 h each night.Throughout the 2019 operating period, specimens identified as FAW from their morphological characteristics (Zhaoet al.2019) were removed from each night’s collection and stored at -20°C for further analysis.The dates and the number of FAW moths were recorded to indicate population dynamics.

No arable lands or host crops of FAW are present on BH,but there are some pine trees and graminaceous weeds.To ascertain whether any FAW moths were produced on BH itself, visual observations and sweep net sampling were carried out daily during the period of the study to detect larvae of this species on any potential wild host plants.

2.2.DNA extraction, amplification, and sequencing

All collected suspected FAW specimens were further confirmed by molecular marker methods.Total genomic DNA of individual specimens was extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany),according to the manufacturer’s recommendations.The DNA preparation was increased to a final volume of 60 μL with distilled water, and stored at -20°C before subsequent analysis.

A mitochondrial (COI) and a nuclear (Tpi) marker were selected for confirming species identity and corresponding host-strain type.Primer pairs CO1-101F (5´-TTCGAGCTGAATTAGGGACTC-3´) and CO1-911R (5´-GATGTAAAATATGCTCGTGT-3´) (811 bp)were used for theCOIregion while primers Tpi282F(5´-GGTGAAATCTCCCCTGCTATG-3´) and Tpi850R(5´-AATTTTATTACCTGCTGTGG-3´) (500 bp) were used to amplify theTpiregion (Nagoshiet al.2017).Primers were synthesized by Sangon Biotech Co., Ltd.(Shanghai,China).PCRs for each gene segment were completed with each specific primer pair in a 25-μL reaction using 2×GoldStar Master Mix (CWBIO, Beijing, China) as previously described (Sunet al.2021).The thermocycling program comprised 95°C (10 min), followed by 35 cycles at 95°C(1 min), 55°C (30 s), 72°C (45 s), and a final extension of 72°C for 5 min.The PCR products were detected by 2%agarose gel electrophoresis, and products with the correct target size were sent to Beijing Genomic Institute (Beijing,China) for sequencing in both directions.

All of the obtained assembled sequences were subjected to molecular identification using the online BLAST search program (http://www.ncbi.nlm.nih.gov/BLAST).Multiple sequence alignments were performed using DNAMAN 6.0 with default gap penalty parameters of gap opening 10 and extension 0.2.

2.3.Stable isotope analysis

The stable carbon isotope(13C/12C) composition offers a powerful means for characterizing the diets of animals(Grahamet al.2010), and recently the approach has been validated and successfully deployed in a number of insects including FAW (Wassenaar and Hobson 1998; Gouldet al.2002; Nagoshiet al.2007).The13C/12C content is commonly reported as δ13C.For insects, only wing tissue is needed to measure the δ13C (Ponsardet al.2004; Jiaoet al.2019).All of the trapped FAW moths had their wings excised with dissection scissors, and the wings were sent to the Stable Isotope Mass Spectrometry Facility, Chinese Academy of Forestry (Beijing, China) for stable isotope analysis.

Samples were analyzed using a Flash Elemental Analyzer (EA1112HT; Thermo Scientific, Bremen, Germany)integratedviaa ConFlo IV system with a DELTA V Advantage Isotope Ratio Mass Spectrometer (Thermo Scientific,Bremen, Germany), following the protocol of Nagoshiet al.(2007).The results are reported in δ13C in units per mill (‰)relative to the Pee Dee Belemnite (PDB) standard.Measurement precision is estimated at ±0.1‰.

2.4.Simulation of backward trajectories

For migratory insect pests, identifying the origin of immigrants is important for understanding their migratory behavior, and is a vital step for pest management as it could enable early pest detection and inform management actions before pests arrive into crops (Hopkinson and Soroka 2010).A three-dimensional backward trajectory analysis (Wuet al.2019a, b) was performed to determine the migration routes and sources of the sampled individuals.Backward trajectories for dates on which FAW was caught were calculated with meteorological data as input; the latter were obtained from a simulation model, Weather Research and Forecasting (WRF) (Skamarocket al.2008), which has been successfully applied to study the migration of various insect species including FAW (Huet al.2013; Wuet al.2019a, b).

In the present study, trajectories were simulated every hour from 19:00 to 06:00 the next morning, which is consistent with the operating time of the searchlight trap on the island, and using a series of initial heights:250, 500,750, 1 000 and 1 250 m above mean sea level (Wolfet al.1990; Mitchellet al.1991).BH was set as the end location.As FAW has strong self-powered flight capacity, a constant airspeed of 4.5 m s-1and a heading offset (difference between insect heading and wind displacement direction) of 30 degree to the right of wind were added to the downwind vector (Wolfet al.1995; Westbrook 2008).The maximum flight duration was set to 12 h also following previous (North American) research (Westbrooket al.2016).

The endpoints of the backward trajectories falling into a large body of water were removed and the locations of simulated endpoints (where FAW took off at 18:00,Westbrooket al.2016) were selected as potential source areas.

3.Results

3.1.Molecular identification and phylogenetic analysis

A total of 142 suspected FAW moths, identified by morphological criteria, were obtained from the searchlight trap samples.All of the BLAST results revealed that theCOI-analyzed sample indeed belonged to FAW, supported by 99-100% similarity in sequence data and coverage.All individuals are identified as “corn-strain” based onTpigene.A totoal of 10 individuals out of the 142 showed variations with A/T or G/A at P174/175 loci.

3.2.Migration dynamics of FAW

No FAW larvae were found on the island during daily field investigations, despite some weeds that were potential hosts being available.The FAW adults trapped by the searchlight trap must therefore have been immigrants from the mainland, which is at least 40-60 km away from the trapping site.

As shown in Fig.1, the number of FAW trap captures was zero until 6 September 2019.Daily catches increased from mid-September to mid-October, and then dropped back to low levels.A short peak occurred on 22-23 October,and then the moths were trapped in low numbers until 3 November.

3.3.lsotopic differentiation of moths from C4 and C3 plants

The δ13C in an adult’s wings can accurately reflect a moth’s larval diet.More specifically, a δ13C value of less than -20.0‰ indicates development on a C3 plant, and a value of more than -15.0‰ as having fed on a C4 plant;an intermediate value does not allow identification of the C3/C4 type of the host plants (Gouldet al.2002; Nagoshiet al.2007).

As shown in Fig.2, δ13C values from wings of FAW moths caught in the BH trap ranged from -31 to -10‰ across all analyzed samples.The great majority (95.07%, 135/142) fell in the -14 - (-10‰) range, and were categorized as having developed on C4 plants, while 3.52% (5/142) specimens,with a δ13C value lower than -20‰, were derived from C3 host plants.In addition, there were two specimens with an intermediate value, indicating that the larvae had fed on both C3 and C4 plants.

Fig.1 Nightly catches of Spodoptera frugiperda moths in the searchlight trap at Beihuang Island, Shandong Province, China in 2019.

Fig.2 The frequency of stable carbon isotope content (δ13C) for a sample of 142 wings captured in a vertical pointing searchlight trap at Beihuang Island, Shandong Province, China.

Fig.3 Potential source sites of trapped Spodoptera frugiperda migrants at Beihuang Island, Shandong Province, China during 1 Sep-31 October 2019.A, endpoints of backward trajectories of trapped moth in potential sites.B, the percentage of the total trajectories that ended in each region.AH, Anhui; HA, Henan;HB, Hebei; JL, Jilin; JS, Jiangsu; LN, Liaoning; NMG, Inner Mongolia; SD, Shandong; TJ, Tianjin.

3.4.Backward trajectory analysis

As shown in Fig.3, northern China (i.e., Hebei, Henan,Jilin, Liaoning and Shandong provinces) was identified as a potential source area for the immigrants.Specifically,the majority of endpoints of backward trajectories were distributed in the Shandong Province (53.34%) and Liaoning Province (37.23%), areas that are not very far from the island, with a few endpoints scattered in more northern areas(Jilin Province).The endpoints of backward trajectories were located at an average distance of 257±169 (mean±SD) km from the trapping site, with the farthest at 909 km.

4.Discussion

BH island, in the center of the Bohai Strait, is an ideal site for investigating insect migration in northern China because:(1) it is a long distance from the mainland; and(2) there is no fresh water and no arable land (Fu X Wet al.2014; Fu X Yet al.2014a, b).Over the past two decades, overseas migration behavior of numerous insect species across multiple orders has been elucidated by our research group at the site (Guoet al.2020).In the present study, searchlight trap monitoring was carried out at this isolated site throughout 2019 (April-October) to monitor population dynamics of the newly-invasive FAW.The number of specimens of the species caught provided direct evidence that FAW moths migrate across the Bohai Gulf,and strongly supports our previous hypothesis concerning the invasion path in northern China (Wuet al.2019a, b).We expect that the pest will establish a regular pattern of seasonal migration across the sea during the forthcoming years, similar to that of FAW populations in North America(Johnson 1987; Westbrooket al.2016; Jianget al.2018).The current data reports on the initial invasion; consistent observations from searchlight trapping, or other techniques such as radar observation, will be needed to determine the pattern of recurring migration as it develops.

Some differences between the “corn-strain” and “rice-strain”concerning pesticide resistance, pheromone composition,and feeding and mating behaviors have been reported(Whitfordet al.1988; Pashleyet al.1995; Veenstraet al.1995; Adamczyket al.1997; Grootet al.2008; Unbehendet al.2013; Ingberet al.2018).Hence, determining strain type of the infesting population could potentially be helpful for the development of appropriate pest management practices.The results of our molecular-marker analyses showed that only the “corn-strain” was present in the BH sample, which is consistent with strain identification in other areas of China, as well as with field investigations showing that FAW populations in China preferentially occur on maize plants (Wuet al.2019a).It is noteworthy that the heterozygous polymorphisms of the two strains were detected in several specimens, as has been the case in molecular strain identification in other areas of China (Jinget al.2019; Zhanget al.2019).

The results of the stable carbon isotope analyses showed that the majority of specimens (95.07%) exhibited a C4 signature, providing additional evidence that this species is a migrant, as there are no major candidate C4 host plants on BH.Additionally, combining data on dates of captured moths by searchlight trapping (September-October) and the strain identification results, we conclude that maize is likely to be the primary C4 source for these moths, because it is a preferred host of the “corn-strain” and large plantings were present around the Bohai region.Taken together, the results suggest that the light-captured FAW were migrants whose larvae primarily developed on corn grown in nearby mainland agricultural areas.

The backward trajectories for FAW immigrants presented here indicate that FAW can move distances of 250 km overnight.It thus appears capable of expanding its range into the maize-cropping areas of Northeast China (especially Liaoning Province), and both molecular (Tpi) and stableisotope results for the migrant population indicate that maize is a suitable host.Hence, despite the fact that Northeast China regions have not been reported being invaded by the FAW population in 2019, it is high likely that these maize-cropping areas would be infested by the pest during the growing season, which is also in good agreement with previous prediction of migratory routes of the invasive FAW in China that FAW emigrants from the Yangtze River Valley reached northern China and pose a high risk to the Northeast Spring Region (Wuet al.2019a; Liet al.2020).Based on previous investigation of migratory behavior of this species (Nagoshiet al.2012; Westbrooket al.2016),the moth is of tropical origin and cannot survive over winter in temperate zones, and thus would re-infest cropping areas at higher latitudes on an annual basis in the summer(and presumably south again the following autumn) (Earlyet al.2018; Liet al.2020).Herein, the southward (return)migration of FAW was supported by our light trap catch data, however, the northward-moving spring migrants have not been detected in our monitoring site perhaps because 2019 is the first invasion year and the population of moths in 2019 was low.As described above, consistent observations are therefore still needed to fully understand the seasonal movements of this species in northern China.Nevertheless,as the northeastern provinces are the major producers of maize in China, the potential range expansion of migratory FAW populations represents a great threat to food security,and authorities in this region need to prepare for the likely impact of this pest.

5.Conclusion

The combination of searchlight trapping, carbon isotope analysis, and backward trajectory analysis on a small isolated island in the center of the Bohai Strait throughout 2019 provided direct evidence that FAW moths can migrate across the Bohai Gulf waters, and that this pest threatens to expand its range into the agriculturally important region of Northeast China where it poses a significant threat to food security.The species identification was determined using both morphology and molecular methods, and only the“corn-strain” that preferentially infests maize and sorghum was found among the migrants.These findings provide baseline information on the occurrence of this species in northern China, which will help in the development of more effective regional management strategies for dealing with it.However, to fully understand the population movements of the pest, as these develop post-invasion, continued monitoring will be needed for some years.Searchlight trapping is effective, but other techniques such as radar observation, would provide additional information of potential value for pest management.

Acknowledgements

This study was funded by the National Natural Science Foundation of China (31901873 and 31727901), the Central Public-interest Scientific Institution Basal Research Fund,China (CAAS-ZDRW202007).

Declaration of competing interest

The authors declare that they have no conflict of interest.