Abstract The redbanded stink bug Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae) is an invasive stink bug species in the United States. First documented as a soybean pest in Louisiana in the year 2000, this species continues to spread in the Mid-South region of the United States. We designed laboratory and field studies to investigate supercooling points, lethal exposure time (LT), critical thermal minimum (CTmin), and winter mortality of this species. The mean supercooling points (SCP) ± SE of adult field collected P. guildinii ranged from −8.3 ± 0.2°C (highest) in March to −11.0 ± 0.2°C (lowest) in January. Significant differences in SCP occurred over the months and between sexes with significant interactions between month and sex. The CTmin was significantly different between adults and nymphs (third, fourth, and fifth instars combined). LT50 and LT90 were evaluated at subzero temperatures of 0°C, −2°C, and −5°C. There were significant differences in LT50 and LT90 among the temperature treatments. Winter survival significantly differed between the two study years and decreased with progression of winter months. Redbanded stink bug, Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae), is a stink bug species found in the tropic and the semi-tropic regions of South and North America (Panizzi and Slansky 1985, McPherson et al. 1993, McPherson and McPherson 2000). P. guildinii was reported from the United States as early as 1960s. However, this species was not considered economically important (Panizzi and Slansky 1985, McPherson et al. 1993, McPherson and McPherson 2000). In 2000, P. guildinii was recognized as a major insect pest of soybean in Louisiana (Baldwin 2004). Subsequently, it has emerged as a serious stink bug pest species (Temple et al. 2013a). Piezodorus guildinii is more damaging to soybean compared to other stink bug species (Depieri and Panizzi 2011). In addition, it is less susceptible to commonly used insecticides compared to other species of stink bug (Temple et al. 2013b). Piezodorus guildinii is the dominant stink bug species in Louisiana soybean, comprising 54% of the species (Temple et al. 2013a). It is established as a major stink bug in southeastern Texas soybean as well (Vyavhare et al. 2014). Current P. guildinii range is distributed along the rice belt of Texas (Vyavhare et al. 2014), throughout Louisiana (Temple et al. 2013a), central Mississippi (Catchot 2009), southern Arkansas (Smith et al. 2009), and southeast Missouri (Tindall and Fothergill 2011). These reports clearly indicate there is a large-scale geographic expansion of P. guildinii populations in recent years. Many biotic as well as abiotic factors influence the colonization, establishment, and expansion of an insect species in a given geographic range (Sakai et al. 2001, Parmesan 2006, Stotter and Terblanche 2009). Thermal tolerance, predominantly low temperatures in winter, is a critical determinant of the geographic range expansion of many insects (Bale 1991b). Though P. guildinii was the third most prevalent stink bug species in soybean in 2009 in southeastern Missouri, no P. guildinii were found in 2010 (Tindall and Fothergill 2011). These episodes of expansion and extirpation of P. guildinii populations in a particular area may be due to fluctuation in climatic conditions (Baur and Baldwin 2006) and low winter temperatures may be limiting its survivorship in northern states (Tindall and Fothergill 2011). Several studies have shown that winter mortality is the major factor that limits the distribution of southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae) (Kiritani 1966, Jones and Sullivan 1981), another neotropical pest. Cold severity and exposure time affect insect physiology and behaviors (Koštál et al. 2007). A prerequisite for an insect species to establish in a geographic area is that adequate numbers of individuals survive periods of winter (Bale 1996). Cold tolerance ability enables insect species to endure cold and consequently build populations rapidly after winter (Denlinger 1991). There are three central mechanisms of cold tolerance in insects based on their physiological response to extended exposure to cold temperatures (Salt 1961, Baust and Rojas 1985, Bale 1993, Zachariassen and Kristiansen 2000, Denlinger and Lee 2010). The first category is ‘freeze-tolerant insects’ that can sustain ice formation outside the body cell. The second category is ‘freeze-intolerant insects’ that evade fatal temperatures by depressing the freezing point of watery solutions inside their own body (Sømme 1982, Baust and Rojas 1985, Zachariassen 1985). Finally, the third category is ‘chill-intolerant insects’ that die before freezing (Bale 1987, 1993; Lee 2010). Cold tolerance is the capability of an insect to endure short or extended exposure time to low temperatures (Salt 1961). Insect cold tolerance studies can help in determining northern distribution limits, relative abundance, and voltinism of the insect species (Bale 1991a, Denlinger and Lee 2010). Evaluation of insect cold tolerance includes measuring of supercooling ability and examination of insect survival at low temperatures (Sømme 1982, Leather et al. 1992). Supercooling ability is a phenomenon in which water and watery solution continue at unfrozen state below the melting point (Bale 1987). Supercooling point determination is a simple procedure that gives the lowest fatal temperature point that freeze-intolerant insects can survive exposure to (Carrillo et al. 2004). A second common index to measure insect cold tolerance is the measure of time–temperature effect on insect mortality (Bale 1996, Denlinger and Lee 2010). Insect survival at colder temperature is dependent on intensity of exposed cold temperature and exposure time. Lethal exposure time (LT) at different subzero temperature has been used to assess cold tolerance in insects (Watanabe 2002). The low-temperature threshold of chill coma onset or critical thermal minimum (CTmin) at which insect enters into reversible state of neuromuscular dysfunction is also used as a measure of cold resistance by insects (Terblanche et al. 2007, Findsen et al. 2014). Winter survival is another good indicator of insect cold tolerance evaluated by observing winter mortality under field conditions (Elsey 1993, Watanabe 2002). The cold tolerance of P. guildinii is an important trait that would allow it to establish in northern parts of the United States, despite its tropical origin. Similarly, low-temperature endurance is an essential feature for insect populations in overwintering survival and is highly relevant in developing pest management strategies (Bale 1991a). Thus, in this study, we studied the cold tolerance and supercooling capacity of P. guildinii. The objectives were to determine (1) the supercooling capacity and seasonal variation in supercooling capacity of the redbanded stink bug, (2) the chill coma temperature (CTmin) of the redbanded stink bug, (3) the lethal exposure time (LT) of the redbanded stink bug at subzero temperatures, and (4) the winter survival of the redbanded stink bug under field conditions. Materials and Methods Measurement of Supercooling Points Adult redbanded stink bugs were collected monthly from May 2015 to April 2016 using a standard (0.38 m diameter) sweep net from the Ben Hur Research Farm (30º 22ʹ12.2ʺN, 91º10ʹ11.6ʺW), Baton Rouge, Louisiana. Insects were collected from soybean during the growing season and from white clover when soybean was absent. Insects were observed for any injuries and only healthy and active individuals were sexed and weighed before use in experiments. All experiments to calculate the supercooling points were conducted within 48 h of insect captivity. Insects were kept at room temperature and no food or water was provided during this period as food and water consumption may alter supercooling points (Salt 1958, Baust and Rojas 1985,). Supercooling points for each individual insect were measured through surface contact thermocouple thermometry. Each insect was placed in 2 ml plastic tube (Thermo Scientific, Nalgene). A small amount (<50 mg) of high-vacuum grease (DOW CORNING, Dow Corning Corporation, Midland, MI) was applied at the base of the tube to immobilize the insect. A thermocouple was attached to the abdomen of each individual immobilized in the tube using the high vacuum grease. A type T (copper/constantan) thermocouple (24-gauge teflon wrap, 0.91-m long, Teflon-coated, DATAQ Instruments, Akron, OH) was used in all experiments. The insect-thermocouple arrangements were retained in a Nalgene Cyro 1°C freezing container (Cat No. 5100-0001). The freezing container was filled with 250 ml of isopropanol alcohol and placed in a −20°C freezer, allowing the insects to cool at the rate of approximately 1°C/min. Temperatures were logged every 0.5 s through a multichannel data logger (DATAQ Instruments, Model DI-1000TC-Y). The data logger used in this experiment was a four-channel data logger that allowed recording supercooling points for four insects in each trial; we conducted 25 trials per month. The SCP was defined as the lowest temperature attained before a sharp rebound visible on the thermal curve owing to the release of the latent heat of freezing. Seasonal changes in supercooling points of field collected P. guildinii were measured throughout the year from May 2015 to April 2016. In each month, supercooling points for 50 male and 50 female adult insects were determined, except in December of 2015 in which 33 male and 33 female insects were used because of the rarity of insects in the field. The monthly change in supercooling points and effect of month, sex, and their interactions were analyzed with analysis of variance (ANOVA). Tukey’s studentized range test (honestly significant difference, HSD) was performed to make multiple comparisons at P < 0.05 (PROC Mixed, SAS Institute 2016). Measurement of Chill Coma Temperature (CTmin) Chill coma temperature is the critical temperature at which insect movement completely stops due to neuromuscular dysfunction resulting from cold temperature (Hazell and Bale 2011, Sinclair et al. 2015). Chill coma temperature was measured for adult (female = 21, male = 21) and nymph P. guildinii (n = 36). The adult P. guildinii used in this experiment were 1-mo old and the nymphs consisted of pooled third, fourth, and fifth instars. These insects were from a laboratory colony maintained at 25°C, RH 45%, 14:10 (L: D) h. The thermoelectric temperature controller (Model no: TC-720, TE Technology INC, MI) that provide desired heating or cooling rate to the Peltier thermoelectric device (Model no: CP-200HTTT, TE Technology INC) was used to induce chill coma. Each insect was placed on its dorsal side and immobilized using vacuum grease on the Peltier plate such that insect was still free to move their legs and antennae. The Peltier plate was cooled at the rate of 0.5ºC/min from the initial plate temperature of 25ºC. Two thermocouples (type-K) were used to measure the temperature, one at the junction of plate and insect body touching the plate and one at the top of the insect body. Each insect was directly observed during the entire cooling process. The chill coma temperature was determined for each insect as the temperature at which there was complete arrest of legs and antennae movement. The average of the two recorder temperatures at the body-plate junction and upper body of the insect was used as the chill coma temperature. The effect of sex on adult as well as stage of P. guildinii on chill coma temperature was analyzed separately with ANOVA. The Tukey’s Studentized Range test (HSD) was performed for separating means at P < .05 (PROC GLM, SAS Institute 2016). Determination of the LT of Redbanded Stink Bug at Different Subzero Temperatures Adult P. guildinii were collected from soybean fields at Ben Hur Research Farm from late planted soybean in the month of October of 2013 and 2014 as described earlier. Collected insects were kept in the laboratory for 12 h at room temperature and provided green beans, raw peanut, and water in a rearing container. Insects were observed for any kind of physical injuries and only insects that were active and in good physical shape were used in the experiment. The critical exposure time LT50, (time required to kill the 50% of the test population) and LT90 (time required to kill the 90% of the test population) for P. guildinii was determined at three subzero temperatures. Based on supercooling points and preliminary tests, 0°C, −2°C, and −5°C were chosen. Adult P. guildinii were exposed to these temperatures for 2–108 h depending upon the temperature chosen. For each exposure time, 30 adult insects were placed individually in 20-ml glass vials without any food and these vials were then incubated in a low-temperature incubator (Precision model no: 815, Thermo Scientific at different temperatures. Control samples of 20 adult individuals were held individually in 20-ml glass vials without any food at room temperature for each subzero temperature treatment. Three trials were conducted for each subzero temperature treatment. After exposure, insects were held at room temperature for 24 h before assessing mortality. Insects that failed to show coordinate movement were consider dead. LT50 and LT90 were calculated through probit analysis (PROC PROBIT, SAS Institute 2016). Winter Survival of Redbanded Stink Bug in Field Conditions This study was carried out during the winters of 2014 and 2015 at Ben Hur Research Farm. Adult P. guildinii were collected from late planted soybean in the last week of October. Small ground cages were made out of PVC pipe measuring 15 cm in diameter and 12 cm in height (Fig. 1). The lower end of the pipe was dug into the ground about 5 cm, whereas the top end was covered with fine fiberglass screen wire mesh of size 24 × 24 cm secured by a 15.24 cm inside diameter galvanized full clamp. Thirty cages with six adults in each cage (three males and three females) were deployed on the last week of October around the edges of the field within mixtures of grasses and clover, which are potential overwintering sites for P. guildinii (Smaniotto and Panizzi 2015, Zerbino et al. 2015). Ten cages were randomly chosen to be destructively sampled on the first week of each month of each year starting in December to February to determine survival of P. guildinii during winter field conditions. Cages were taken to the laboratory where soil and foliage were manually processed and examined thoroughly for live P. guildinii. Numbers of live and dead P. guildinii and sex of those insects were recorded. A logistic regression was used to predict the mortality of P. guildinii using years, months, and sex (PROC LOGISTIC, SAS institute 2016). Fig. 1. View largeDownload slide Ground cages made out of PVC pipe and fiberglass screen wire mesh for the study of winter survival of P. guildinii at Ben Hur Research Farm, Baton Rouge, Louisiana. Fig. 1. View largeDownload slide Ground cages made out of PVC pipe and fiberglass screen wire mesh for the study of winter survival of P. guildinii at Ben Hur Research Farm, Baton Rouge, Louisiana. In this analysis, odds ratios were used to compare the effect of each factor with their respective reference. The odds ratio represents the odds of mortality occurring for a given factor compared to odds of mortality occurring at their respective reference factor. The coldest recorded soil temperature from 10-cm depth and air temperature and total chill hours (numbers of hours when the temperature is less than 7.2°C) for the months of November, December, and January in 2013 through 2014 and 2014 through 2015 from Ben Hur Research Farm were obtained from Louisiana Agriclimatic Information System website (LSU AgCenter 2016). Results Seasonal Variation in Supercooling Ability of Adult Redbanded Stink Bug There were significant differences in the observed mean SCP of P. guildinii over time (F = 14.08; df = 11, 1142; P < .0001). The mean SCP (± SE) of adult P. guildinii ranged from the highest (−8.3 ± 0.2°C) in March to the lowest (−11.0 ± 0.2°C) in January (Table 1). The mean SCP of adult P. guildinii for the month of March was significantly higher than the months of May, August, September, November, December, and January. Similarly, the mean SCPs from April, June, and July were significantly higher than winter months of November, December, and January. In addition, mean SCPs from May, August, and October were significantly higher than SCPs in January (Table 1). Table 1. Mean supercooling points (°C ± SE) of field collected P. guildinii from Ben Hur Research Farm, Baton Rouge, Louisiana, in 2015–2016 Month n Means ± SE March 100 −8.3 ± 0.2a July 100 −8.9 ± 0.2ab April 100 −9.2 ± 0.2abc June 100 −9.3 ± 0.2abc September 100 −9.4 ± 0.2bc October 100 −9.9 ± 0.2bcd August 100 −10.0 ± 0.2cd May 100 −10.0 ± 0.2cd November 100 −10.4 ± 0.2de February 100 −10.6 ± 0.2de December 66 −10.9 ± 0.3de January 100 −11.0 ± 0.2e Month n Means ± SE March 100 −8.3 ± 0.2a July 100 −8.9 ± 0.2ab April 100 −9.2 ± 0.2abc June 100 −9.3 ± 0.2abc September 100 −9.4 ± 0.2bc October 100 −9.9 ± 0.2bcd August 100 −10.0 ± 0.2cd May 100 −10.0 ± 0.2cd November 100 −10.4 ± 0.2de February 100 −10.6 ± 0.2de December 66 −10.9 ± 0.3de January 100 −11.0 ± 0.2e Means followed by different letters are significantly different by Tukey’s studentized range (HSD) test with P < .05. View Large Effect of Sex on Supercooling Ability of Adult Redbanded Stink Bug There was significant effect of sex on the mean SCPs of adult P. guildinii (F = 4.04; df = 1, 1142; P = .0446). Combined over all seasons, the mean SCP of male adult P. guildinii was significantly lower than female adult P. guildinii (Table 2). In addition, there was a significant interactions between sex and month (F = 2.25; df =11, 1142; P = .0106) for SCPs of adult P. guildinii (Table 3). The mean SCP of female P. guildinii from March, June, and September was significantly higher than male P. guildinii from December and January. Similarly, the mean SCP of male P. guildinii from March, April, and July was significantly higher than female P. guildinii from December, January, and February (Table 3). Table 2. The mean SCP (°C ± SE) and mean weight (g ± SE) of P. guildinii combined over the year Sex SCP Weight Female −9.71 ± 0.09b 0.063 ± 0.001a Male −9.96 ± 0.09a 0.051 ± 0.001b Sex SCP Weight Female −9.71 ± 0.09b 0.063 ± 0.001a Male −9.96 ± 0.09a 0.051 ± 0.001b Means followed by different letters are significantly different by Tukey’s Studentized Range (HSD) test with P < .05. View Large Table 3. Mean supercooling points (°C ± SE) of field collected male and female P. guildinii from Ben Hur Research Farm, Baton Rouge, Louisiana, in 2015–2016 Month n (female) Means ± SE (female) n (male) Means ± SE (male) March 50 −7.6 ± 0.3a 50 −8.9 ± 0.3abc September 50 −8.9 ± 0.3abc 50 −9.8 ± 0.3bcdef June 50 −9.0 ± 0.3abcd 50 −9.5 ± 0.3bcde July 50 −9.4 ± 0.3bcde 50 −8.5 ± 0.3ab April 50 −9.4 ± 0.3bcde 50 −8.9 ± 0.3abc October 50 −9.5 ± 0.3bcde 50 −10.3 ± 0.3cdef May 50 −9.8 ± 0.3bcdef 50 −10.1 ± 0.3cdef August 50 −9.9 ± 0.3bcdef 50 −10.1 ± 0.3cdef November 50 −10.0 ± 0.3cdef 50 −10.9 ± 0.3ef February 50 −10.8 ± 0.3ef 50 −10.4 ± 0.3cdef December 33 −10.8 ± 0.4def 33 −11.1 ± 0.4ef January 50 −11.2 ± 0.3f 50 −10.9 ± 0.3ef Month n (female) Means ± SE (female) n (male) Means ± SE (male) March 50 −7.6 ± 0.3a 50 −8.9 ± 0.3abc September 50 −8.9 ± 0.3abc 50 −9.8 ± 0.3bcdef June 50 −9.0 ± 0.3abcd 50 −9.5 ± 0.3bcde July 50 −9.4 ± 0.3bcde 50 −8.5 ± 0.3ab April 50 −9.4 ± 0.3bcde 50 −8.9 ± 0.3abc October 50 −9.5 ± 0.3bcde 50 −10.3 ± 0.3cdef May 50 −9.8 ± 0.3bcdef 50 −10.1 ± 0.3cdef August 50 −9.9 ± 0.3bcdef 50 −10.1 ± 0.3cdef November 50 −10.0 ± 0.3cdef 50 −10.9 ± 0.3ef February 50 −10.8 ± 0.3ef 50 −10.4 ± 0.3cdef December 33 −10.8 ± 0.4def 33 −11.1 ± 0.4ef January 50 −11.2 ± 0.3f 50 −10.9 ± 0.3ef Means followed by different lower case letters in the column and different upper case letters in the rows are significantly different by Tukey’s studentized range (HSD) test with P < .05. View Large Chill Coma Temperature (CTmin) The chill coma temperature (CTmin) for female and male P. guildinii ranged from 6.9 to 9.5°C and 7.3 to 9.6°C, respectively. The mean chill coma temperature for male P. guildinii was slightly higher (8.3 ± 0.2°C) than female P. guildinii (7.9 ± 0.2°C). The chill coma temperature range for P. guildinii nymph was 7.2–11.0°C. The mean chill coma temperature between adult (8.2 ± 0.14°C) and nymph (8.9 ± 0.15°C) P. guildinii was significantly different (F = 10.35; df =1, 70; P = .0020). Lethal Exposure Time The LT to kill 50% and 90% of adult P. guildinii populations at subzero temperatures 0, −2, and −5°C were determined (Table 4). LT50 for 0, −2, and −5°C was 53.4, 37.4, and 6.8 h, respectively. Likewise, the LT90 at 0, −2, and −5°C was 75.4, 56.81, and 10.36 h, respectively. There were significant differences in LT for tested subzero temperatures as indicated by the 95% fiducial limits (Table 4). Table 4. LT (h) required for 50% and 90% mortality of adult P. guildinii from Ben Hur Research Farm, Baton Rouge, Louisiana, to subzero temperatures Temperature(°C) n LT50 (h) 95% FL LT90 (h) 95% FL Slope ± SE χ2 0 810 53.41 51.18–55.64 75.44 72.32–79.17 0.058 ± 0.003 23.27 −2 720 37.41 35.93–40.00 56.81 53.93–60.30 0.068 ± 0.004 15.69 −5 630 6.77 6.25–7.29 10.36 9.59–11.24 0.363 ± 0.031 32.51 Temperature(°C) n LT50 (h) 95% FL LT90 (h) 95% FL Slope ± SE χ2 0 810 53.41 51.18–55.64 75.44 72.32–79.17 0.058 ± 0.003 23.27 −2 720 37.41 35.93–40.00 56.81 53.93–60.30 0.068 ± 0.004 15.69 −5 630 6.77 6.25–7.29 10.36 9.59–11.24 0.363 ± 0.031 32.51 Nonoverlapping 95% fiducial limits (FL) shows significant differences in LT at given temperature. View Large Winter Survival in Field Conditions There were significant differences (χ2 = 23.74; P < .01) in the mortality of P. guildinii between the winter months of 2013 through 2014 and 2014 through 2015 (Table 5). Likewise, the mortality was significantly different (χ2 = 75.84; P < .01) among the winter months tested (Table 5). However, differences were not detected (χ2 = 2.75; P < .11) in the mortality between sex (Table 5). The estimated odds ratio of mortality of P. guildinii for the winter of 2013 through 2014 was 3.61, which suggests that the mortality of P. guildinii was 3.61 times higher than in the winter of 2014 through 2015. Similarly, when compare within winter months, the mortality of P. guildinii was 0.04 and 0.11 times lower in November and December than January, respectively. Table 5. Odds ratio estimates (95%CI) for the winter mortality of adult P. guildinii from Ben Hur Research Farm, Baton Rouge, Louisiana Effect Odds ratio 95% CI P-value Year 2013–2014 3.61 2.15–6.06 <0.01 2014–2015 (Reference) Month Nov. 0.04 0.023–0.092 <0.01 Dec. 0.11 0.062–0.227 0.02 Jan. (Reference) Effect Odds ratio 95% CI P-value Year 2013–2014 3.61 2.15–6.06 <0.01 2014–2015 (Reference) Month Nov. 0.04 0.023–0.092 <0.01 Dec. 0.11 0.062–0.227 0.02 Jan. (Reference) View Large Discussion Piezodorus guildinii is a stink bug species of tropical origin that is expanding its range into cooler regions probably due to warmer winters (Panizzi 2015). This study provides the firsthand informations on the cold tolerance of this species. This study shows that there is seasonal variation in the supercooling capacity of field populations of P. guildinii. The SCPs for P. guildinii were higher in summer months and decreased with the progression of fall and winter months. Depression of SCPs as insects enter fall and winter months has been documented in a number of insects (Baust 1972, Bale 1980, Sømme 1982, Carrillo et al. 2005, Hou et al. 2009, Bale and Hayward 2010, Cira et al. 2016). This observation is also an evident that P. guildinii undergo seasonal acclamation possibly due to change in temperature regimes in their surroundings. The lowest mean SCP of P. guildinii (−11.0 ± 0.2°C) and the lowest minimum recorded temperature from the sampling location (−2.2°C) both occurred in January (Fig. 2). The cold tolerance study on the southern green stink bug from South Carolina has reported that the SCP of this species is approximately −11°C (Elsey 1993). The comparable cold tolerance study of the invasive brown marmorated stink bug, Halymorpha halys (Stål) (Hemiptera: Pentatomidae) from Minnesota and Virginia has also reported significant difference in the mean SCP over the seasons (Cira et al. 2016). The mean SCP of brown marmorated stink bug occurring in those regions was −9.43°C in summer, −15.4°C in fall, and −16.11°C in winter (Cira et al. 2016), which is lower than P. guildinii and the southern green stink bug. Fig. 2. View largeDownload slide Seasonal changes in minimum and maximum air temperatures at Ben Hur Research Farm, Baton Rouge, Louisiana from May 2015 to April 2016 (Source: Louisiana Agriclimatic Information System). Fig. 2. View largeDownload slide Seasonal changes in minimum and maximum air temperatures at Ben Hur Research Farm, Baton Rouge, Louisiana from May 2015 to April 2016 (Source: Louisiana Agriclimatic Information System). In many insects, depression of SCP is associated with accumulation of cryoprotectants such as glycerol and antifreeze compounds like alcohols and sugars (Sømme 1982, Tauber et al. 1986, Bale 1987, Atapour and Moharramipour 2009, Hou et al. 2009). We found lowest SCP in the month of January and the highest in the month of March (Table.1). Similar observations were reported for the Asiatic rice borer larva, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), with lowest SCP in winter and highest in March (Hou et al. 2009). It is also reported that body water content of Asiatic rice borer larva significantly increased and the hemolymph glycerol significantly plummeted from winter to March (Hou et al. 2009). Therefore, comparable phenomena may also be associated with adult P. guildinii. It is shown that in fall and winter, adult P. guildinii has reduced body size, underdeveloped reproductive organs, and excess lipid reserves (Zerbino et al. 2015). It is possible that the lowest mean SCP for field acclimated population of P. guildinii observed in the month of January may be due to higher lipid reserves. On the other hand, the lowest mean SCP in the month of March may have resulted from the depletion of lipid reserve as insects become active during early spring. We found that there were significant differences in SCP between sexes. The difference between sexes may be due to difference in body weights. As males weigh less than females and are usually smaller, differences in total body water content might affect SCP (Salt 1956, Miller 1969, Sømme 1982). Based on this study, P. guildinii cold tolerance strategies appears to be freeze intolerant because no survival was found once the SCP was reached and the insect body froze. The SCP represents the lowest temperature at which a freeze-intolerant insect could stay alive without body freezing, but insect mortality can occur well above the SCP due to chilling injuries as well (Bale 1987). For this reason, we cannot rule out the possibilities of P. guildinii as chill intolerant, which needs further investigations. Nonlethal low temperatures can also have profound effects on insects that can directly affect their foraging, reproduction, and defense against predation and parasitism (Hazell and Bale 2011). The CTmin represents nonlethal temperatures at which an insect enters into a chill coma characterized by a reversible state of complete lack of movement (Hazell and Bale 2011, MacMillan and Sinclair 2011). Our data indicate that adult P. guildinii has lower CTmin than nymphs do. Difference in thermal tolerance has been reported in insects based on their life stages (Bowler and Terblanche 2008, Marais et al. 2009). Lower CTmin of adult P. guildinii means adults may be more capable to endure the effects of nonlethal cold temperature compared to nymphs. In our experiment, CTmin was evaluated only from laboratory colony. It has been reported that acclimatization to colder temperatures can increase critical thermal limits in insects (Colhoun 1960). Therefore, further experiment with field acclimatized insect populations will elucidate the importance of CTmin in overwintering biology of P. guildinii. The LT to kill 50% of P. guildinii at −5°C in our experiment is 6.8 h, which is more than five times shorter than the diapausing adult southern green stink bug as reported by Elsey (1993). Similarly, the LT50 at 0°C is just over 2 d and the LT50 at 2°C of a day and half. The results from LT indicates that the mortality of P. guildinii collected in the fall season is rapid under subzero temperatures. Our study shows that winter survival of P. guildinii under field conditions depends upon the severity of low temperature during the winter. The predicted mortality of P. guildinii was 3.61 times higher in the winter of 2013 through 2014 compare to the winter of 2014 through 2015. The unusually severe cold spell of the winter of 2013 through 2014 in Louisiana was responsible for the higher mortality. The lowest recorded air temperature was −7.7°C in the month of January and this observation was comparable to the LT study in the laboratory conditions. The lowest soil temperature of 3.3°C also occurred during January of 2013 through 2014 (Table 6). However, during the mild winter of 2014 through 2015, the predicated mortality of the P. guildinii was lower. The lowest air temperature recorded was −2.2°C in November and lowest soil temperature recorded was 6.6°C in January. The mortality significantly increased as the winter months progressed. This may be the result of accumulation of chilling injuries, as exposure to chill hours increased with time. However, other stressors like starvation and desiccation may also directly affect the winter survival (Koštál et al. 2007, Terblanche et al. 2011). The incidents of rise and successive fall of P. guildinii populations may best be explained by the severity of winter temperatures occurring in those regions. The favorable winter conditions in this time of climate warming may be a probable cause of its recent range expansions. As for future studies, we would suggest to use these data in temperature models to predict the range of expansion of this insect with temperatures predicted for future years. Table 6. Temperature records from Ben Hur Research Farm, Baton Rouge, Louisiana (Climatic data source: Louisiana Agriclimatic Information System) Year Month Chill hoursa Coldest recorded soil temp (°C) (10 cm) Coldest recorded air temp (°C) 2013–2014 Nov. 148.3 8.3 −3.3 Dec. 267.9 8.3 −1.6 Jan. 362.9 3.3 −7.7 2014–2015 Nov. 160.5 10.0 −2.2 Dec. 82.4 10.5 1.1 Jan. 318.4 6.6 −1.1 Year Month Chill hoursa Coldest recorded soil temp (°C) (10 cm) Coldest recorded air temp (°C) 2013–2014 Nov. 148.3 8.3 −3.3 Dec. 267.9 8.3 −1.6 Jan. 362.9 3.3 −7.7 2014–2015 Nov. 160.5 10.0 −2.2 Dec. 82.4 10.5 1.1 Jan. 318.4 6.6 −1.1 aChill hours = number of hours exposed to temperature <7.2°C. View Large In conclusion, this study provides the first insights into the cold tolerance of P. guildinii in the United States. Information on cold tolerance of P. guildinii is critical for understanding their possible geographic range, relative abundance, and seasonal activity and distribution patterns. This information will form the basis for developing a model to forecast overwintering survival and potential pest population numbers. Further investigation on ecophysiological bases of the P. guildinii will elucidate the underlying mechanism of cold tolerance. Acknowledgments The authors would like to thank members of the soybean entomology laboratory at LSU with their help in insect collections. We greatly appreciated Dr. James P. Geaghan for his help with data analysis. Appreciation is also expressed to Dr. T. E. Reagan and other reviewers of our paper. This study was partially funded by the Louisiana Soybean and Grain Research and Promotion Board, the Louisiana State University Agricultural Center and the United Soybean Board Award No. 1420-532-5652. 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