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

Abstract Fall armyworm,Spodoptera frugiperda (J.E.Smith,1797),a crop pest native to tropical and subtropical regions of America,has invaded and spread into most regions in China,posing a severe threat to China’s agriculture.The cold hardiness directly determines its geographic distribution through adapting to winter temperatures of different regions.Here,we measured supercooling points and lethal time (LT) at low temperatures of S. frugiperda.The supercooling points for developmental stages in increasing order were:adults (–15.05°C)＜pupae (–13.25°C)＜prepupae (–10.50°C)＜larvae (–9.03°C).Among eggs and 1st to 4th instar larvae,the 99% lethal time (LT99) was the highest for 4th instar larvae,with 99% of larvae dying after 18.59 d at 2°C,58.72 d at 7°C,and 66.28 d at 13°C.LT99 was the lowest for eggs with LT99 of 5.33 d at 2°C,9.28 d at 7°C,and 12.97 d at 13°C.This study provides an understanding of overwintering regions of S. frugiperda in China,which will be helpful for population forecasting and management.

Keywords:Spodoptera frugiperda,supercooling point,overwinter,China

1.Introduction

The fall armyworm (FAW),Spodopterafrugiperda(J.E.Smith,1797),native to tropical and subtropical regions of American continent (Sparks 1979),is one of the most damaging crop pests worldwide.This pest has rapidly spread through the Old World and was first captured in China on 11 December 2018 (Sunet al.2021).Since then,it has become one of the major invasive pests in China,seriously threatening agricultural production.FAW consists of two strains,the “corn-strain” and the “rice-strain”.The FAW population that invaded China was the “corn-strain”(Zhanget al.2019a),and there is a high similarity in theCOIsequence between the Chinese FAW samples and those from Florida (Zhanget al.2019b).FAW has a wide host range of more than 350 plant species (Montezanoet al.2018),but principally attacks corn,sugarcane and sorghum in China.Larvae in peanut fields feed on growing points and young organs,with damage rates of 80% at the seedling stage and 62% at flowering (Heet al.2020).Yanget al.(2020a,b) reported that the damage rates of FAW were 30–100% in wheat fields and 9.33–100% in barley fields.FAW may also be a threat to tobacco when its population density is high (Xuet al.2019).

The migratory behavior of FAW plays a very important role in its invasions.In January 2016,major outbreaks of FAW were reported in southwestern Nigeria and Ghana,then shortly after in Benin,northern Nigeria and in Sao Tomé and Togo (Goergenet al.2016).Just one or several nights are needed to complete the flight from West Africa to southern Sahara (Earlyet al.2018).As of 28 September 2017,28 sub-Saharan African countries had confirmed the presence of FAW which had a devastating impact on corn production,with a further nine countries were suspecting or awaiting official confirmation of its presence (Dayet al.2017).In May 2018,FAW was discovered in Karnataka in southwestern India (Sharanabasappaet al.2018),and by late 2018,FAW outbreaks had been found in other Asian countries,such as Thailand,Sri Lanka,Bangladesh,Myanmar (Guoet al.2018) and China (Sunet al.2021).Wuet al.(2019a,b) accurately predicted the migration routes of FAW from Myanmar to China (Yunnan and Guangxi were the main areas) and its migration routes from tropical and southern subtropical zones of China in spring and summer(mainly toward the northeast).As of 8 October 2019,the FAW had invaded 26 provinces (autonomous regions,municipalities) in China.

The ability of the FAW to migrate long distances,hundreds or even thousands of kilometers on high-altitude winds over several successive nights (Westbrooket al.2016),has enabled it to spread rapidly in Africa and China.This ability is also one of the reasons why it is one of the most damaging crop pests in the world (Roseet al.1975; Westbrooket al.2016; Dayet al.2017).This migratory ability is particularly necessary for species that do not diapause and therefore have a limited temperature range for overwintering.Since FAW lacks any diapause mechanism,it can only survive in tropical and subtropical areas during the winter in America and elsewhere.In spring,FAW from tropical regions can migrate to places where it can breed continuously when the temperature is suitable and host plants are available(Dayet al.2017).In the United States,it can only survive in southern Florida and Texas during the winter where hosts are continually available and temperatures are rarely below 50°F (10°C) (Sparks 1979).During the next growing season,the insects disperse again throughout the eastern and central United States and into southern Canada (Roseet al.1975; Mitchellet al.1991).

Mechanisms for insect cold hardiness include chilling and cold-acclimation,supercooling to avoid freezing,and freezing tolerance.Usually insects living at low altitudes and latitudes are less cold-tolerant than those at high altitudes and latitudes.As a native of tropical and subtropical America,FAW tolerates high temperatures (Sparks 1979);thus,its cold tolerance should determine its geographic distribution and ability to adapt to winter temperatures in different regions.When Foster and Cherry (1987) exposed all life stages of FAW to low temperature for 3 h,the egg was the most tolerant stage,with 30% survival at–10°C.In China,Xieet al.(2019) reported that the mortality of 1st–3rd instar larvae was 100% when the average temperature was below 10°C for 8–10 days.Although larvae of FAW can harm overwintering wheat and barley in China (Yanget al.2020a,b),whether the larvae can overwinter on the crops has been unknown.Here we measured the supercooling points(SCPs) of different development stages and the survival abilities of eggs and larvae at different low temperatures.The results will provide a scientific basis for predicting the timing of the occurrence of the FAW and regions where it can overwinter in China.

2.Materials and methods

2.1.Insect rearing

We used DH2019 population collected in January 2019 from Dehong Autonomous Prefecture,Yunnan Province in China.The population was reared on an artificial diet for about three generations before the testing.The artificial diet was molten agar containing mainly wheat bran and soybean powders.The diet was mixed thoroughly with a rod,allowed to cool and harden,then cut into small pieces.One FAW larva was placed into each well of 24-well plates that contained pieces of diet and reared in the insect chamber with controlled environment of (26±1)°C,(60±10)% RH and a 14 h:10 h (L:D) photoperiod.

2.2.Determination of SCP and freezing point (FP)

We determined the SCP and FP of different development stages (2nd–6th instar larvae,prepupae,different ages of pupae,and female and male adults) using SCP determination equipment (SUN-V intelligent insect supercooling point tester; Pengcheng Electronic Technology Center of Beijing,China).The heat-sensitive detector was inserted into a 1.5-mL tube in direct contact with the cuticle of the insect and fixed in place with cotton around it.The tube was immersed in a refrigerated ethanol bath that was cooled at a rate of 1°C min–1(DW-FL35 ultra low temperature freezing storage tank; Zhongke Meiling Low Temperature Technology Co.,Ltd.,Hefei,China).The temperature of the tested individual decreased with the environmental temperature.SCP as the lowest temperature before insect body temperature starts to increase as ice forms,and FP is the temperature at which ice crystals formed and endotherm and exotherm were in balance.For each treatment,three replicates of 32 individuals were measured.

2.3.Assays of survivability at low temperature

Eggs,1st,2nd,3rd and 4th instar larvae were subjected to 2°C,7°C (HYC-391 low temperature medical refrigerator;Qingdao Haier Co.,Ltd.,Qingdao,China),and 13°C(MGC-450HP climate chamber; Shanghai Yiheng Scientific Instruments Co.,Ltd.,China).We checked mortality every day until they were all dead,then calculated the exposure time required to kill 50,90 and 99% of the larvae (LT50,LT90and LT99).Larvae were considered dead if they did not move in a coordinated manner when touched several times with a small soft brush.

2.4.Data analyses

The SCPs and FPs of different developmental stages of FAW were examined using one-way analysis of variance(ANOVA),and means were separated using LSD test(P＜0.05).The LT values at low temperatures were analyzed by probabilistic value analysis.The logarithm value of insect survival time at low temperatures is linearly related to the probability value of mortality (Egeret al.1982).All analyses were performed using SPSS Statistics 26.0 (SPSS,Chicago,IL,USA).

3.Results

3.1.SCP and FP of FAW population

The SCP of different developmental stages (Fig.1; Appendix A) differed significantly (F3,577=270.406,P＜0.001).The SCP of adults was significantly lower than for pupae (P＜0.001),and that of pupae was significantly lower than for prepupae(P＜0.001),and that of the prepupae was significantly lower than for larvae (P＜0.001).The difference in SCPs among 1-,3-,5-and 7-day pupae was not significant (F3,228=0.334,P=0.801).But significant differences were found among different larval stages (F4,261=35.791,P＜0.001).For the SCP of 2nd instar larvae,which was significantly lower than for 3rd instar larvae (P＜0.05),that of 3rd instar larvae was significantly lower than for 4th instar larvae (P＜0.05),that of 4th instar larvae was significantly lower than for 5th and 6th instar larvae (5th instar larvae:P＜0.001,6th instar larvae:P＜0.001),but the difference between that of 5th and 6th instar larvae was not significant (P=0.328).The difference in SCPs for males and females was also not significant (P=0.239).

The FP of different developmental stages (Fig.1;Appendix A) differed significantly (F3,577=34.784,P＜0.001).The mean FP of adults was significantly lower than for pupae(P＜0.001),and that of pupae was significantly lower than for larvae (P＜0.001),but not for prepupae (P=0.138).The mean FP of the prepupae was lower than for larvae,but also not significant (P=0.613).Significant differences in FPs were found among larval stages (F4,261=20.216,P＜0.001); that of 2nd instar larvae were significantly lower than for 3rd instar larvae (P＜0.001),that of 3rd instar larvae were significantly lower than for 4th instar larvae (P＜0.05).However,the difference between the FP of 4th instar larvae and that of 5th was not significant (P＜0.065),and the difference between that of 5th instar larvae and that of 6th instar larvae was also not significant (P=0.934).The difference in FPs among 1-,3-,5-and 7-day pupae was significant (F3,228=3.209,P＜0.05); that of 1-day pupae was significantly lower than for 7-day pupae (P＜0.05),but was not for 3-day pupae(P=0.266) and 5-day pupae (P=0.226).The FP of females was significantly lower than for males (P＜0.05).

3.2.Cold tolerance

The LT99for eggs and 1st–4th instar larvae was 5.33–18.59 d at 2°C,9.28–58.72 d at 7°C and 12.97–66.28 d at 13°C,respectively (Table 1; Fig.2).The results showed that the survival at low temperature of 3rd and 4th instar larvae was greater than for eggs,1st and 2nd instar larvae although their SCPs were higher.The 1st,2nd,3rd,and 4th instar larvae did not complete generation development at 2,7,or 13°C.

4.Discussion

In China,migratory insect pests move toward northern regions from their overwintering areas in southern regions in the spring; therefore,understanding the overwintering biology of an insect pest is important.In general,the northern overwintering boundaries of migratory insect pests are based on the cold hardiness (Table 2).The northern overwintering boundaries ofMythimnaseparataandSpodopteraexiguawere both located at about 0°C isotherm of average daily temperature in January (Liet al.1993; Jianget al.2001;Zhenget al.2017).Wuet al.(1997) reported that–10°C isotherm of average daily temperature in January was the northern overwintering boundary ofHelicoverpaarmigera.AndOstrinianubilaliscan overwinter in the northeastern China (Liuet al.2005).However the overwintering region of FAW in China has not been confirmed.In the United States,it can only survive in southernmost regions of Florida and Texas where temperatures below 50°F (10°C) are rare during the winter (Sparks 1979); its northern overwintering boundary is located at about 28°N.

Fig.1 Supercooling points and freezing points measured for Spodoptera frugiperda at different developmental stages.The left panel is supercooling points,and the right is freezing points.Different capital letters below error bars indicate a significant difference among means (P＜0.05).Bar indicates SE (n=96).

Table 1 Mean LT50,LT90 and LT99 of Spodoperta frugiperda eggs and different instar larvae at low temperatures

In the present study,the SCPs of adults (–15.05°C) and pupae (–13.25°C) of FAW were significantly lower compared to those of prepupae (–10.50°C) and larvae (–10.50°C).The SCP of pupae (–13.25°C) of FAW was significantly higher than that ofM.separata(–16.26°C) (Table 2).However,the SCP is not enough for the measurement of cold tolerance and must be supplemented with cold exposure experiments(Leeet al.1987; Joneset al.2008).We found that among eggs and 1st to 4th instar larval stages,the LT99was the highest for 4th instar larvae,the lowest for eggs.However,Foster and Cherry (1987) reported that among all life stages of FAW from Florida,eggs were the most tolerant,with 30% survival at–10°C,perhaps because they tested the extremely low temperatures below 0°C.In addition,the survival time we found here was longer than that reported in fields in China (Xieet al.2019),likely due to the many other factors that can influence mortality in the field.

The FAW does not diapause at any developmental stages (Sparks 1979).Heet al.(2019a) reported that the developmental threshold temperature of eggs,larvae,pupae and adults was 10.27,11.10,12.20 and 11.34°C,respectively.Here,we found that,at 7°C,the LT99of egg,1st instar larvae and 2nd instar larvae was shorter than 31 d (January),but the LT99of 3rd and 4th instar larvae was longer than 31 d.According to the data above and climatic regionalization of China,we preliminary proposed the annual breeding area for FAW includes southern area of the 10°C isotherm of average daily temperature in January:Guangdong,Guangxi,Hainan,Taiwan,Fujian,the southern and central regions of Yunnan.The areas that between the 10°C isotherm and 7°C isotherm of average daily temperature in January are the overwintering region for FAW; the northern boundary from east to west is southern Zhejiang,southern and central Jiangxi,southern Hunan,southern Guizhou,and northern Yunnan.Northern area of the 7°C isotherm of average daily temperature in January is the immigration area.On the basis of the LT99for the various stages at different low temperatures,we speculate that at 7°C (northern overwintering limit),the eggs and young larvae will quickly die,while some older instar larvae and pupae might survive much longer.

The northern overwintering boundary ofM.separatais located at the 0°C isotherm of average daily temperature in January (Liet al.1993).Southern Guangdong,Guangxi,Fujian,Hunan and Jiangxi are the important sources ofM.separatain China,whereM.separatacan produce 6–8 generations a year; they then migrate to the north in February (Jianget al.2014),and produce 5–6 generations a year in the Yangtze River area,4–5 generations a year in the Yellow-huai River Basin.Mythimna separataproduce 3–4 generations a year after they enter the northern China plains in April and May,and 2–3 generations a year after they migrate to the northeastern China plains in May and June (Jianget al.2014).The northern overwintering boundary of FAW is located at about 7°C isotherm of average daily temperature in January,with the same number of generations described forM.separate; however,the timing is slightly later.The tropical and subtropical areas of Yunnan,Guangxi,Guangdong,Hainan,Fujian and Taiwan are the important sources for FAW in China,where they produce 6–8 generations a year.FAW produce 5–6 generations a year after they migrate into the southern Yangtze River areas in March,4–5 generations a year after they arrive the Yellow-huai River Basin in April and May,and 3–4 generations a year after moving to the northern China plains in May and June; populations from these plains migrate to the northeastern China plains in June and July and produce 2–3 generations a year there (Wuet al.2020).

Fig.2 Mean survival of Spodoptera frugiperda eggs and different instar larvae at 2°C (A),7°C (B) and 13°C (C).Bar indicates SE (n=96).

The overwintering ability of insects directly influences population levels the next year.Determining their resistance to cold is thus important not only for understanding the ecological mechanism and evolution of a pest,but also for forecasting and integrated management of pest.Cold hardiness studies of different geographical populations of cotton bollworm in China significantly improved forecasting accuracy and provided a foundation for establishing a regional warning system (Wuet al.1997).Management strategies to decrease the number of pests in early spring can also be proposed based on the overwintering habit of a pest.The mortality rates of overwintering pupae of cotton bollworm were up to 80–90% in fields that had been cultivated and irrigated in winter,2-fold lower than in fields that were not cultivated and irrigated (Guo 1998).

Here,we predicted that FAW can overwinter in southern parts of subtropical zones in China,but this prediction needs to be proved by field investigation.The overwintering populations of FAW can be effectively reduced by cultivation,irrigation and straw removal in the overwintering regions.The peak emergence of adults in the next spring when the climate is suitable can also be predicted by field monitoring,so that timely management strategies can decrease the number of migrating adults.Potential immigration regions can be predicted based on migration routes (Wuet al.2019a) and flight capacity of FAW in China (Geet al.2019).The dynamics of female oviposition and larval hatching in immigration regions can also be predicted according to ovarian development gradation,reproduction potential prediction of females and the major axis length of testis of males (Heet al.2019b; Zhaoet al.2019).The prevention and control of FAW in overwintering regions is a critical first step toward managing FAW populations for the entire year.Thus,knowledge on cold hardiness and overwintering regions of FAW is invaluable for designing management strategies to effectively reduce damage below the economic injury level.To adequately understand the cold hardiness of FAW and confirm its overwintering regions in China,we also need to understand how other factors,such as light,humidity,rainfall,host plants and drought,indirectly and directly influence insect overwintering,and natural enemies.

5.Conclusion

The cold hardiness of the FAW decides its overwintering region.This study on cold hardiness of the invasive FAW population in China indicates that the survivability of pupae and older larvae is much greater than that of eggs and younger larvae.Based on the SCPs of developmental stages and LT99of FAW at low temperatures and the climatic regionalization of China,we estimate that FAW may survive in some southern areas of the subtropical zone in China during the winter season.Our results provide a foundation for designing a forecasting system and management strategies to control this most serious pest in China.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31727901 and 31901873) and the National Key Research and Development Program of China(2019YFD0300101).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendixassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm