Study on the Ecology, Biology and Ethology of the Invasive Species Corythucha arcuata Say, 1832 (Heteroptera: Tingidae), a Danger to Quercus spp. in the Climatic Conditions of the City of Sibiu, Romania

: This scientiﬁc paper examines the impact of climate change on the spread and survival of the invasive species Corythucha arcuata Say, 1832 (oak lace bug, or OLB) in Romania. The OLB is native to the Eastern USA and Canada, and in its native habitat, it coexists with minimal impact to the preferred host plant species Quercus . However, in Europe, the OLB in high density causes early defoliation, slowing tree growth. As Quercus spp. is a keystone species, the impact of the OLB on European forests is signiﬁcant. This paper aims to address several gaps in knowledge about the OLB by presenting ﬁndings from a study conducted by the Lucian Blaga University of Sibiu (ULBS) in Romania. The study identiﬁed the species’ chronology of spread, ecology, ethology and biology of the species, in addition to foliage classiﬁcation to determine the extent of attack. The study found a direct link between the temperature and the speed of the generational lifecycle. The ﬁndings support the thesis that climate change is enabling the healthy, rapid density growth of OLB, which is a danger to the Quercus spp. Further research is needed in the area of the thermal tolerance of the OLB, and the research conducted by the ULBS represents the ﬁrst documented research into the thermal constant of the insect. The paper concludes that further research must be in the direction to understand how and where these insects survive during winter to ﬁnd future management measures and identify chemical or biological methods as solutions to eradicate and stop the expansion of the invasion of the OLB, and the potential consequences for the Quercus sp.


The Chronology of OLB Spread in Europe
When first identifying OLB, the researchers sought more information about its status in Europe. There was no easy to go to place that brought the different sightings and chronology together. Extensive time-consuming desk research was undertaken. We hope the chronological information and map below and the density of reported species sighting will save other researchers time ( Figure 1). OLB was first reported in Europe in northern Italy (Lombardy and Piedmont regions) in May 2000, on Quercus robur L., Q. pubescens Willd. and supposed hybrids of Q. robur L. and Q. petrea (Matt.) Liebl. [19,20].
In 2002, OLB extended to the south of Switzerland, where the species was detected in April and July 2002 near Pura (Malcantone). In the summer of 2003, it was found on fallen oak leaves in a mixed forest near Chiasso, southern Switzerland [21]. Researchers hypothesized that OLB arrived in the Swiss Canton of Ticino in 2001 or 2002, immigrating from Lombardy (Italy) [22].
In 2003, OLB was recorded in Turkey for the first time in the province of Bolu and then in the provinces of Düzce, Zonguldak, Sakarya, Kocaeli, Eskişehir, Ankara, Çankırı and Bilecik [23,24]. Subsequent research in Turkey led to the conclusion that the species has infected 28,000 km 2 of territory [9,25] and is now listed as a permanently monitored invasive species [26,27].
In 2005, it was detected in Iran [28]. In 2005, OLB was reported in Iran and West Azerbaijan [28].
In Poland, it was announced for the first time in 2009, and since then, 27 more alerts have been reported [29].
In 2012, July and September, OLB was reported in Bulgaria for the first time on the leaves of Quercus robur L. in Plovdiv and mixed stands formed by Q. robur L. and Q. cerris L. in the vicinity of Zlatidolvill near the city of Simeonovgrad in southern Bulgaria. Most likely, OLB entered Bulgaria from the Asian part of Turkey, where it is widespread [30]. In just five years after the first registration, OLB invaded most of Bulgaria, where the attack reached approximately 85% of the infested trees [31].
By 2013, the pest expanded its European range, entering Hungary [32]. It was detected for the first time in Debrecen, Eastern Hungary [33], and later it spread throughout Hungary [34][35][36]. Currently, the species is being monitored, and from data collected in different research, 11 subspecies of oak have been reported as host plants. Based on these findings, experts conclude the only thing that will restrict the expansion of OLB in Europe and Asia is a lack of host trees [37].
Moreover, in 2013, in Croatia, OLB was confirmed in five locations on elm stands (Ulmus minor Mill.) [38]. In 2014, OLB expanded to over 30,000 ha through the Spačva basin, the largest and most valuable complex oak forest (Quercus robur L.) in the EU. By 2015, it has spread throughout the Spačva basin, attacking 60,000 ha of forest [39].
In 2015, OLB entered Russia and was found in the Krasnodar city park [24]. Satellite images of the oak forests confirmed the OLB spread into the North-West Caucasus [43] and extended to the south of the European part of Russia [44][45][46][47]. In 2015, the first report of OLB in Romania was published.
In 2016 (Autumn), OLB was reported in south-east Slovenia on leaves of Quercus robur L. in an oak forest in the village of Zakot, near Brezice. Specialists hypothesized the spread came from Croatia, where it was previously reported in the Luznica Castle park near Zapresic, just 14 km from the park in Slovenia. The area had good rail and car traffic routes along the northern edge of the infested forest. In 2017, the spread of the species was reported in the Krakovo oak forest [48]. More recently, monitoring studies have been set up across Slovenia through the Life Artemis project, which engages citizens to send the coordinates of the places where they observed the pest on the trees [49].
In 2018, saw the first record of OLB in Slovakia in June, on leaves of Quercus cerris near the village of Mužla, close to the Danube in southern Slovakia and in August neař Cičarovece, in eastern Slovakia. Researchers confirm the hypothesis of the spread came from Hungary, where a strong infestation was reported in several areas of the country [56].
In 2018, OLB was discovered in the central area of Bosnia in two localities. Specialists indicated the possibility of its spread throughout Bosnia and Herzegovina in the coming years [54].
In 2019, Austria recorded OLB in September in south-east Styria and south Burgenland in 21 locations in the districts of Hartberg-Fürstenfeld, Leibnitz, south-east Styria and Güssing and Jennersdorf. Infestation intensity varied widely between 1% and 95% [57]. Moreover, in 2019 was reported in North Macedonia [58] and in the Czech Republic [59].
Since its accidental introduction around the year 2000, OLB has spread rapidly over a wide geographical area and is now found in over 20 other European countries [60]. The latest reports highlight the OLB species affects ALL Eurasian oak species and will also feed on other woody plant species [36,61]. The most frequently attacked trees are the Oak species of Quercus robur L., Q. frainetto Ten., Q. petraea (Matt.) Liebl. and Q. cerris L. [50].
In March 2001, this invasive species was recognized as a potential threat to the European oak and was declared by the European and Mediterranean Organization for Plant Protection on the list of alerts an EPPO [50].
In March 2001, this invasive species was recognized as a potential threat to the European oak and was declared by the European and Mediterranean Organization for Plant Protection on the list of alerts an EPPO [50].
The 1% damage intensity has no impact on the tree's leaves and corresponds to occasional adult populations of up to 10 per host plant. A large attack that can reach 100% reflects a significant number of individuals from several generations that overlap in all three development phases (eggs, nymphs of different generations and adults). Adults and nymphs feed by perforation in the leaf epidermis, resulting in chlorotic patches, where the leaf saps are damaged by the degradation of chlorophyll pigments. In a large attack, all the tree's leaves are damaged.
In 2021, in Sibiu, we identified OLB on a sessile oak tree in the inner courtyard of the Natural History Museum. In 2022, under observation, we confirmed the expansion in all areas with oak and sessile oak species. Our local concern related to the necessity of issuing a warning in an attempt to prevent the spread of OLB in the county's extensive forest ecosystems. the leaf saps are damaged by the degradation of chlorophyll pigments. In a large attack, all the tree s leaves are damaged.
In 2021, in Sibiu, we identified OLB on a sessile oak tree in the inner courtyard of the Natural History Museum. In 2022, under observation, we confirmed the expansion in all areas with oak and sessile oak species. Our local concern related to the necessity of issuing a warning in an attempt to prevent the spread of OLB in the county s extensive forest ecosystems.

Materials and Methods
Sibiu is located in southern Transylvania, in the center of Romania. The city is crossed by the Cibin river watercourse, bordered to the north by Mureș County, to the south by Vâlcea and Argeș counties, to the east by Brașov County and to the west by Alba and Hunedoara counties. Sibiu city is in a temperate (moderate) continental climatic area. Secondary microclimatic thermal influences exist due to the ground wind direction, influenced by relief factors caused by a depression area and the Carpathian and Cindrel mountain ranges that surround the city to the south and southwest. Annual average precipitation is 662 mm, with minimum values in February (26.7 mm) and maximum in June (113 mm) [71]. The average annual temperature is 8.9 °C [71]. The climate, relief and soil structure create favorable conditions for rich flora and fauna, with a high-density oak and sessile oak forest area that climbs from the city to the hills and slopes of the Cindrel Mountains. In exploring the OLB spread in Sibiu, we started monitoring the species in the

Materials and Methods
Sibiu is located in southern Transylvania, in the center of Romania. The city is crossed by the Cibin river watercourse, bordered to the north by Mures , County, to the south by Vâlcea and Arges , counties, to the east by Bras , ov County and to the west by Alba and Hunedoara counties. Sibiu city is in a temperate (moderate) continental climatic area. Secondary microclimatic thermal influences exist due to the ground wind direction, influenced by relief factors caused by a depression area and the Carpathian and Cindrel mountain ranges that surround the city to the south and southwest. Annual average precipitation is 662 mm, with minimum values in February (26.7 mm) and maximum in June (113 mm) [71]. The average annual temperature is 8.9 • C [71]. The climate, relief and soil structure create favorable conditions for rich flora and fauna, with a high-density oak and sessile oak forest area that climbs from the city to the hills and slopes of the Cindrel Mountains. In exploring the OLB spread in Sibiu, we started monitoring the species in the summer of 2021 and throughout 2022, studying its biology, ecology and, especially, its ethology. We tracked down the OLB locations and, based on data collected from the presence of host trees in the observational sites, we created a map representing the spread of the species (Figure 3). Through our ULBS study we believe our findings to be of scientific importance as we identify novel elements relating to the species' tolerance to minimum winter temperatures and the number of annual generations that are impacted by the meteorological environment (temperature, humidity and wind direction).
During August to October 2021 and February to October 2022, we conducted 18 site observations (P 1 -P 18 ), all located in the urban area of Sibiu, not only in isolated stands but also in parks (Sub Arini park-4 sites, Astra and Cetăt , ii parks-2 sites), green areas (Natural History Museum of Sibiu, Turismului street, Cârlova street, S , tefan Cel Mare street, Ovidiu street-3 sites, city's cemetery, Sibiu's Zoo) from the districts of S , trand, Hipodrom, Gus , terit , a and Turnis , or, and on trees of Dumbrava Sibiului forest's edge ( Figure 3).From these observation points, leaves infected with the species at all three stages of development of the insect were sampled and are described in detail in Section 3.1. Microscopic images on sessile oak leaves from all observation points are shown at all three stages of development (egg, nymph, and adult) in Figures 4-8, while results on the intensity of the insect's attack in the 18 observation points are shown in Section 3.2 in Figures 9-13. Data collected included the attack intensity, indicating the percentage of the attacked leaves (Figures 9-13). During May-September 2022, we observed OLB adults at distances greater than 500 m from any host tree. This involved detailed monitoring over a two-year period at the 18 sites detailed in the Study Area. We then followed a methodology studying the morphology of the insect and their extent of attack. Signs of OLB presence appear on the underside of the leaves, with yellowish to brownish spots, with the presence of adults, nymphs, or dark excreta recognized as additional signs [9,72,73].    To determine the attack severity, we use the degree of leaf discoloration, with the benchmark scale being the normal unaffected leaf color. The degree of attack was determined by viewing both sides of leaves affected by OLB, ensuring we excluded from the analysis discoloration produced by other variables (attack by other insect species, diseases, or other physiological causes such as dryness) (Figures 9-13). Roughly 150 leaves were analyzed at each observation station (P 1 -P 18  Throughout the two years, ethological studies of the species were conducted, documenting the sum of temperatures at each developmental stage and the number of generations. As an invasive species, we witnessed the natural adult spread in all 18 observational districts during the summer of 2022. Adults were carried by air currents, on the clothing of locals, or by cars.

Legend
The development and evolution of OLB in Sibiu's climatic conditions included aspects related to zoogeographical propagation. OLB spread was characterized by a complex set of factors, the most important of which were thermal and precipitation factors.
Three generations were observed between 2021 and 2022, with a single generation being composed of egg stages, five stages of nymphs and development to the adult stage.
To understand better the period of the OLB development cycle and the number of generations in each year, we processed meteorological data (Sibiu Meteorological Station) to determine the following: biological threshold of OLB, thermal constant (K), prolificacy threshold (O), thermal optimum (O 1 ) and upper threshold (T).
The difference (t n − t 0 between the absolute temperature (t n ) and the biological threshold (t 0 ) was used to calculate the effective temperature.
Each developmental stage and each generation required a sum of different effective temperatures, which we calculated using Blunck's developmental equation (1914)(1915)(1916)(1917)(1918)(1919)(1920)(1921)(1922)(1923), Equation (1): where x n -time of development, t 0 -temperature of development. In this equation, K is represented by the effective temperature sum, characteristic of the years 2021 and 2022, necessary for one generation development from the stage of egg to nymph stage, then adult.
To determine the period required for the development of one generation, we calculated the multiplication between the period of development x n and the effective temperature (t n − t 0 ).
OLBs value of thermal constant (K) was represented by the sum of the development stage thermal constants being egg laying, the appearance of nymphs and adults.
The population dynamics during the development and growth period in Sibiu' climatic conditions are represented by the number of generations Equation (2), in 2021 and in 2022, which we correlated to the actual temperatures using the multiplication equation [74].
The aridity index by Martonne was calculated annually and expressed as being the restrictive character of climatic conditions Equation (3)   Additionally, the precipitation amount during the growing season was an important factor in the OLB spread. Abundant August rainy days were one factor that influenced the third-generation (G 3 ) dynamic. In June-July 2021-2022, Sibiu had a water deficiency, which, according to Connor's (1988) research, could affect the population feeding preferences, affirming that OLB does not feed on water-deficient leaves, thereby slowing growth cycles [75].
The monthly maximum, minimum and average temperatures, and the correlated quantity of precipitations in 2021-2022 in the city of Sibiu are listed in the tables below: The lowest temperature months were January and February 2021 and January, Febru  0.12 mm and 0.94 mm, while the average rainfall in the wettest month was 3.08 mm and 3.53 mm.
As we better understood how thermal constant played a key role in the generational cycle development, we also wanted to understand how OLB handles extremely low temperatures and if the average warming of minimum temperature in Europe's climates could be aiding OLB to overwinter and survive.

Results
Of the 18 observational sites (P 1 -P 18 ) (Figure 3), the most affected habitational type by OLB, were the trees from parks (Sub Arini, Cetăt , ii, Astra, city cemetery, Sibiu Zoo) where the extent of attack reached 80%-90% at the end of vegetation period. On the sessile oak tree located in the Natural History Museum yard, the attack was 95% (observation point P 1 as presented in Figure 13), while on other isolated trees the average attack extent was 45% (observation points P 3 , P 6 , P 7 , P 8 , P 13 and P 17 as presented in Figures 9-13), up to 75% (Figures 11-13).
Our observational findings concluded that OLB adults preferred the following species of trees: Q. robur L., Q. polycarpa Schur. And Q. petraea (Matt.) Liebl., which grow under an open canopy. Abundance occurs in areas where the trees were exposed to most light, where the leaf surface was greater, and therefore the level of carbon was higher. As observed in Astra, Sub Arini and Cetăt , ii parks, but also on isolated trees from S , trand, Hipodrom, Gus , terit , a and Turnis , or districts. Abundance density correlates to the leaf water content [76].

Biology of the Species C. arcuata (Say, 1832)
Adults are relatively small, with a body length of 3-4.5 mm and a width of 1-2.5 mm. The head is completely hidden by the pronotal hood, which ends just above the level of the head.
The antennae are long, slender and covered with hairs [2]. The first antennae segment length is at least double that of the second, the third is considerably longer than the first two together with the fourth segment shorter than the third and slightly thicker.
The pronotum is broad and covered with small spines on the outer margins. The wing is broad at the base and the outer margin is strongly reflected anteriorly, with a tumid elevation near the inner anterior margin.
The insect has erected nervures in an irregular pattern on its hemelytra and pronotum [2]. In spring, after wintering, OLB is found on oak leaves immediately after they appear. Adults feed on the host tree young leaf sap, highlighting their ability to overwinter. Both adults and instars (the phase between two periods of molting) feed on leaf sap by stinging the leaf epidermis and extracting the sap from their cellular tissue. Only the stylets (mandibles), which are very thin parts of their mouth, can penetrate the leaf to suck the sap to feed with it [77]. Females lay long, black eggs ( Figure 4A) on the abaxial side of leaves, the number of eggs can vary from 15 up to over 100 eggs per female [78]. According to Bernathinelli [79], the medium number of eggs laid on the trees analyzed in north Italy is around 100 eggs per cluster, and during one vegetative period, 3-4 egg generations were laid. OLB growth and development from egg to adult composed of 5 successive separate stages of instars (molting), which in Sibiu's climatic conditions, took between 4 and 6 weeks [77].
The OLB eggs were embedded deep in the lower side of leaves ( Figure 4A), almost to the operculum, then covered with a brown sticky excretion which rapidly hardens, forming a protective coat. After emerging and drying, the nymphs start to feed, forming clusters near the empty eggshells and adults. If disturbed, they regroup fast. Leaf chlorosis appears because after hatching from the egg clusters, nymphs feeding on the leaf sap begin immediately. Where there are several different nymph stages, feeding is intensive. This leads to complete degradation of chlorophyll pigments.
At the end of 2021, following the field surveys in the months of November, December, January 2022 and February, we observed that OLB overwinters in the adult stage in, on or under bark cracks at a depth of 3-4 cm and displays gregarious behavior even in the hibernation site under the bark, under the fallen leaves on the ground or in the moss on the bark.
In Spring (21 April 2021 and 5 May 2022), only a few adults were identified on leaves (1-3 individuals).
The first generation of adults (G 1 ) was observed at the beginning of May 2021 and at the end of April 2022.
Second generation adults (G 2 ) were observed at the beginning of June 2021 and at the end of July 2022.
Only the adults observed at the end of August developed a third generation (G 3 ).
In 2021-2022, the development time from nymph to adult varied from 16 to 39 days in Sibiu's climate, compared to 13 to 27 days in the Italian region climate [72]. Following the monitoring at the 18 sites (P 1 -P 18 ) and examining the number of eggs on the abaxial side of leaves, we noticed the fact that the OLBs egg is composed of two distinct regions, the basal part, which is smooth, surrounded by an unknown substance secreted by the female and operculum, which is formed by a lip and an apical cap, thus being the most distinctive egg zone.
Not only the edge but also the cap is very porous [78,79]. After counting, we noticed the presence of some groups of eggs, which varied between 7 and 80 eggs, being laid alongside the midrib of leaf (Figures 4-8). In Sibiu, the adults and the nymphs were observed on the abaxial side of leaf while feeding on the host tree, causing numerous dark spots, while the adaxial side of leaf presented chlorotic discolorations areas [9,72]. The first generation of adults (G1) was observed at the beginning of May 2021 and at the end of April 2022.
Second generation adults (G2) were observed at the beginning of June 2021 and at the end of July 2022.
Only the adults observed at the end of August developed a third generation (G3).
In 2021-2022, the development time from nymph to adult varied from 16 to 39 days in Sibiu s climate, compared to 13 to 27 days in the Italian region climate [72]. Following the monitoring at the 18 sites (P1-P18) and examining the number of eggs on the abaxial side of leaves, we noticed the fact that the OLBs egg is composed of two distinct regions, the basal part, which is smooth, surrounded by an unknown substance secreted by the female and operculum, which is formed by a lip and an apical cap, thus being the most distinctive egg zone.
Not only the edge but also the cap is very porous [78,79]. After counting, we noticed the presence of some groups of eggs, which varied between 7 and 80 eggs, being laid alongside the midrib of leaf (Figures 4-8). In Sibiu, the adults and the nymphs were observed on the abaxial side of leaf while feeding on the host tree, causing numerous dark spots, while the adaxial side of leaf presented chlorotic discolorations areas [9,72].              From July through October, all phases of insect development (eggs, nymphs of varying ages and adults) were seen on the undersides of leaves. In the absence of natural limiting conditions, it is recognized that a powerful attack reduces the resilience of host trees, making them susceptible to attack by additional diseases and/or other insect pests [9,30]. From July through October, all phases of insect development (eggs, nymphs of varying ages and adults) were seen on the undersides of leaves. In the absence of natural limiting conditions, it is recognized that a powerful attack reduces the resilience of host trees, making them susceptible to attack by additional diseases and/or other insect pests [9,30].

Ecology of Species C. arcuata (Say, 1832)
Temperature, precipitation and light were ecologically significant physical elements that affected the growth of OLB in the climate of Sibiu.
As with many insects, OLB are poikilothermic creatures, with their body temperature varying in response to the thermal fluctuations of their surroundings [80].
The temperature of hibernating adults dropped significantly during the diapause, which also explains the endurance of adults during the winter of 2021-2022, when the minimum temperature in January was −3.7 °C, in February it was −2.53 °C and in March it was +1.79 °C when they emerged from the diapause. Thermal constants, at temperature values, at which insects undergo drastic changes in biochemical processes, are of special relevance in their life and activity and allow the acquisition of knowledge of favorable and unfavorable thermal development circumstances [74].
For OLB, the thermal constants at which the determined chemical and biochemical processes take place were calculated. Thus, it was discovered that OLB development occurred at the biological development threshold (t0), which was 0.3 °C (in 2021) and 0.1 °C (in 2022) (Equation (4) and Table 3). In Sibiu, OLB enters the hyaline diapause if temperatures fall below these parameters. In the development stage equation, was substituted this temperature value (t0) and derived the thermal constant (K), which had values of 14.2 °C (in 2021) and 9.85 °C (in 2022) (Equation (5)). In Sibiu, above these temperatures, OLB entered summer diapause.
Adult G1 flight occurred in early May 2022. Meteorological circumstances during the 2022 OLB growing season had a major effect, impacting its metabolic activity, abundance rate and dispersion over Sibiu.
The air temperature (minimum, mean and maximum) proved crucial in determining the OLB behavior [81].
The values of the De Martonne aridity index Equation (3)

Ecology of Species C. arcuata (Say, 1832)
Temperature, precipitation and light were ecologically significant physical elements that affected the growth of OLB in the climate of Sibiu.
As with many insects, OLB are poikilothermic creatures, with their body temperature varying in response to the thermal fluctuations of their surroundings [80].
The temperature of hibernating adults dropped significantly during the diapause, which also explains the endurance of adults during the winter of 2021-2022, when the minimum temperature in January was −3.7 • C, in February it was −2.53 • C and in March it was +1.79 • C when they emerged from the diapause. Thermal constants, at temperature values, at which insects undergo drastic changes in biochemical processes, are of special relevance in their life and activity and allow the acquisition of knowledge of favorable and unfavorable thermal development circumstances [74].
For OLB, the thermal constants at which the determined chemical and biochemical processes take place were calculated. Thus, it was discovered that OLB development occurred at the biological development threshold (t 0 ), which was 0.3 • C (in 2021) and 0.1 • C (in 2022) (Equation (4) and Table 3). In Sibiu, OLB enters the hyaline diapause if temperatures fall below these parameters. In the development stage equation, was substituted this temperature value (t 0 ) and derived the thermal constant (K), which had values of 14.2 • C (in 2021) and 9.85 • C (in 2022) (Equation (5)). In Sibiu, above these temperatures, OLB entered summer diapause. Adult G 1 flight occurred in early May 2022. Meteorological circumstances during the 2022 OLB growing season had a major effect, impacting its metabolic activity, abundance rate and dispersion over Sibiu. The air temperature (minimum, mean and maximum) proved crucial in determining the OLB behavior [81].
The values of the De Martonne aridity index Equation (3)

Ethology of the Species C. arcuata (Say, 1832)
Our 2022 studies focused on the attacked trees, where we noticed that the intensity of the attack was far more severe compared to 2021. OLB growth was based on a complex set of physical, chemical, biochemical and biological reactions whose speed varied according to temperature. The degree of attack at 80%-90% was greater in trees at the edge of the Dumbrava Sibiului forest (P 12 ) and the cemetery (P 9 -P 11 ). Trees exposed to full light and with sun exposure were more affected (P 1 , P 13 -P 16 , P 18 ) compared to those inside the stands, with the differences of values reaching 50%. At the observational site (P 2 -P 8 ), the interior trees were 25% attacked. The population dynamics during the vegetation period of 2021-2022 varied according to weather factors, and the climate changes during the day influenced daily activity. Some researchers have shown that the insect density on the leaves may not be associated with increased leaf nutrient concentrations but with the amount of light, higher concentrations of carbon in the leaves (C), lignin and total phenols, and lower levels of cellulose [82].

Ethology of the Species C. arcuata (Say, 1832)
Our 2022 studies focused on the attacked trees, where we noticed that the intensity Our research supports this thesis. At the end of September 2021, according to our observations, OLB wintered in the adult stage, or eggs laid by the third generation. We are questioning the possibility that eggs are overwintered. Because during 2021-2022, we had three overlapped generations (G 1 -G 3 ). More research in 2023 will seek to understand this. To study the OLB ethology under Sibiu's climatic conditions, we analyzed the thermic constants, which are specific to this species (Table 3).
The growth and reproduction occurred at the temperature of the prolificacy threshold (O), and the shortest development time and maximum prolificacy occurred at the thermal optimum (O 1 ), while the biological activity of the species ceased at the level of the upper threshold (T). These values calculated for each year are shown in Table 3.
The minimum temperature at which OLB reacted in the climatic conditions of Sibiu, as well as the average temperatures specific to each generation Equation (4), are presented in Table 4. Because the third generation appeared towards the end of August as the generation that went through diapause as adults, the developmental equations were only computed for the first and second generations.

Ethology of the Species C. arcuata (Say, 1832)
Our 2022 studies focused on the attacked trees, where we noticed that the intensity of the attack was far more severe compared to 2021. OLB growth was based on a complex set of physical, chemical, biochemical and biological reactions whose speed varied according to temperature. The degree of attack at 80%-90% was greater in trees at the edge of the Dumbrava Sibiului forest (P12) and the cemetery (P9-P11). Trees exposed to full light and with sun exposure were more affected (P1, P13-P16, P18) compared to those inside the stands, with the differences of values reaching 50%. At the observational site (P2-P8), the interior trees were 25% attacked. The population dynamics during the vegetation period of 2021-2022 varied according to weather factors, and the climate changes during the day influenced daily activity. Some researchers have shown that the insect density on the leaves may not be associated with increased leaf nutrient concentrations but with the amount of light, higher concentrations of carbon in the leaves (C), lignin and total phenols, and lower levels of cellulose [82].
Our research supports this thesis. At the end of September 2021, according to our observations, OLB wintered in the adult stage, or eggs laid by the third generation. We are questioning the possibility that eggs are overwintered. Because during 2021-2022, we had three overlapped generations (G1-G3). More research in 2023 will seek to understand this. To study the OLB ethology under Sibiu s climatic conditions, we analyzed the thermic constants, which are specific to this species (Table 3).   Table 4. Average temperatures specific to each generation during 2021-2022.

Year 2021/Average Temperature According to Generation
Year 2022/Average Temperature According to Generation t 1 = K 1 x 1 = 765.5 95 = 8.06 for G 1 t 1 = K 1 x 1 = 776.9 110 = 7.06 for G 1 t 2 = K 2 x 2 = 778.9 41 = 18.99 for G 2 t 2 = K 2 x 2 = 771.8 32 = 24.12 for G 2 t 3 = K 3 x 3 = 770.9 33 = 23.36 for G 3 t 3 = K 3 x 3 = 779.3 35 = 22.27 for G 3 The OLB development constants in Sibiu's climatic conditions were calculated using Blunck's evolution equation (1914) elaborated by Săvescu, 1969 [78] Equation (5), calculated by us for each year according to the following correlations: Prolificacy threshold (O): has been represented by the temperature degree t n whereas above these values, the two genders of OLB become fertile, Equation (6). In 2021, the OLB prolificacy threshold was 5.02 • C, while in 2022, it increased to 5.32 • C. Superior threshold of temperature (T): In Sibiu's climatic conditions of 2021-2022, the superior threshold of temperature was constituted by the maximum temperature values above whom the species interrupts its development. The maximum temperature supported by Equation (7)  The multiplication constants C and √ X: The two coordinates of point C, where the multiplication hyperbole and the regression line of the number of generations γ in a year intersect, will be t n and x n Equation (8):  Our field research identified three strong generational lifecycles, with the extent of attack being stronger in 2022 than 2021, suggesting a good overwintering and with the climate conditions of 2022 being beneficial to both rapid lifecycle development through five stages to adult and multiple generations. Other factors do come into play, such as levels of precipitation as dry leaves slow the development cycle and light as OLB prefers sunnier, light-exposed areas more than trees in the middle of the wood with less sun and greater shade. Where OLB has light population numbers and few overlapping generations of nymphs, the density of attack is low. However, where population density is high and several generations of nymph all feed at the same time on the oak leaf sap, such that the attack density is high, defoliation occurs. This long-term will be a problem in the healthy development and survivability of the tree, especially if defoliation occurs early in the season. Our statistical research on thermal constant highlighted a tight correlation between temperature and speed of lifecycle development, see the graph below ( Figure 14).

Discussion
The desk research identified an alarming, explosive growth in OLB range and density in Europe from 2012 onward. Climatic records from the Sibiu Meteorological Office highlight a marked jump in average temperatures and lower average minimum temperatures also from the decade of 2010+, with the warmest records for Sibiu being 2022, 2019 and 2020, respectively. This supports the fact that the climate now in Europe matches the native climatic conditions.
Where the earlier spring, later fall and higher average temperatures with reasonable precipitation levels exist, it is feasible for four generation cycles to fully develop. We witnessed three development cycles in observation points from the Sibiu County area. OLB comes out of its overwintering phase when the average minimum temperature reaches above freezing, i.e., at 0.1c. Feeding begins immediately on the young leaves, which will be rich in sap. As can be seen in the graph, the acceleration of a lifecycle can be less than 16 days from nymph to adult (13 days recorded in Italy). Little is still known about OLBs capability to overwinter and how it achieves this. Further field research is being conducted

Discussion
The desk research identified an alarming, explosive growth in OLB range and density in Europe from 2012 onward. Climatic records from the Sibiu Meteorological Office highlight a marked jump in average temperatures and lower average minimum temperatures also from the decade of 2010+, with the warmest records for Sibiu being 2022, 2019 and 2020, respectively. This supports the fact that the climate now in Europe matches the native climatic conditions.
Where the earlier spring, later fall and higher average temperatures with reasonable precipitation levels exist, it is feasible for four generation cycles to fully develop. We witnessed three development cycles in observation points from the Sibiu County area. OLB comes out of its overwintering phase when the average minimum temperature reaches above freezing, i.e., at 0.1c. Feeding begins immediately on the young leaves, which will be rich in sap. As can be seen in the graph, the acceleration of a lifecycle can be less than 16 days from nymph to adult (13 days recorded in Italy). Little is still known about OLBs capability to overwinter and how it achieves this. Further field research is being conducted in the 2022-2023 winter phase. We do not yet know if both forms of adult and egg survive winter or just the adult. We do not yet know how the adult successfully overwinters.
Are adults form of OLB burying themselves in the soil at a temperature they can survive at, or is the micro-climate of the cracks in the Oak at 3-4 cm sufficient to keep them alive? OLB has few natural predators in Europe. Chemical control has not been entirely successful and biological control has not been tested enough.
Our further research is oriented in 2023 in this direction to test these control methods deeper. The lack of hard wing cases and the delicate "lace wing" like structure means they are more susceptible to extreme cold conditions. However, the marked warming of Europe in the recent decade is a key factor in the establishment of OLB and the explosive growth in recorded sightings.

Changing Climate Leading to Higher Temperatures
According to data from the National Meteorological Administration of Romania (ANMR), the year 2022 was considered the 3rd warmest year in the history of meteorological measurements in Romania, the average annual temperature was 11.77 • C, with a temperature deviation of 1.55 • C from the average period between 1981 and 2010. In the time span 1900-2022, the five warmest years were 2019, 2020, 2022, 2015 and 2007. Additionally, the interval between 2012 and 2022 represents a period of 11 consecutive years with the highest temperatures in Romania (Figures 15 and 16). This information confirms the trend of increasing air temperatures in our country. The climate changes that have led to positive temperature anomalies in Romania between 0.68 • C (2021) and 1.92 • C (2019), reported between 2012 and 2022, correspond to the trends reported globally, but especially in Europe [76,80]. Moreover, from the data provided by ANMR, the time period between 1991 and 2020, considered the current climatic reference period, registers an increase of 0.5 • C between multiannual mean air temperatures in Romania, compared to the time between 1981 and 2010. The largest temperature increases are in the last two decades (2001-2010 and 2011-2020), with frequent daily, monthly, seasonal and annual temperature records being recorded in the last twenty years [83]. Europe in the recent decade is a key factor in the establishment of OLB and the explosive growth in recorded sightings.

Changing Climate Leading to Higher Temperatures
According to data from the National Meteorological Administration of Romania (ANMR), the year 2022 was considered the 3rd warmest year in the history of meteorological measurements in Romania, the average annual temperature was 11.77 °C, with a temperature deviation of 1.55 °C from the average period between 1981 and 2010. In the time span 1900-2022, the five warmest years were 2019, 2020, 2022, 2015 and 2007. Additionally, the interval between 2012 and 2022 represents a period of 11 consecutive years with the highest temperatures in Romania (Figures 15 and 16). This information confirms the trend of increasing air temperatures in our country. The climate changes that have led to positive temperature anomalies in Romania between 0.68 °C (2021) and 1.92 °C (2019), reported between 2012 and 2022, correspond to the trends reported globally, but especially in Europe [76,80]. Moreover, from the data provided by ANMR, the time period between 1991 and 2020, considered the current climatic reference period, registers an increase of 0.5 °C between multiannual mean air temperatures in Romania, compared to the time between 1981 and 2010. The largest temperature increases are in the last two decades (2001-2010 and 2011-2020), with frequent daily, monthly, seasonal and annual temperature records being recorded in the last twenty years [83]. These climatic changes may be the hypothesis of the OLB invasion in our country, as the insect finds climatic conditions similar to its country of origin. The development of the species and the attack of Quercus sp. in the climatic conditions of Sibiu ( Figure 17) and the appearance of the three generations (Table 3)   These climatic changes may be the hypothesis of the OLB invasion in our country, as the insect finds climatic conditions similar to its country of origin. The development of the species and the attack of Quercus sp. in the climatic conditions of Sibiu ( Figure 17) and the appearance of the three generations (Table 3)      Based on these temperatures, it can be observed that the number of days decreased between generations starting with G 1 (95 days in 2021 and 110 days in 2022), G 2 (41 days in 2021 and 32 days in 2022) and G 3 (33 days in 2021 and 35 days in 2022) directly proportional to the temperature increase between May and August ( Figure 17). The distribution of seasonal precipitation amounts in the year 2021-2022 were below climatological averages in the fall, spring and summer seasons, except in winter 2021-2022, when December recorded values above normal.

Conclusions
OLB was first documented in Sibiu in 2021 on an oak tree in the Natural History Museum's courtyard (P 1 ). During 2021 and 2022, this tree's leaves were marked to monitor the number of generations and determine development cycles. Another 17 places (P2-P18) were examined, where the same parameters relating to the development and generation numbers, as well as attack extent, which ranged from 25% to 95%, were monitored ( Figure 3). The species of trees attacked in Sibiu (Quercus robur L., Q. petraea (Matt.) Liebl., Q. polycarpa Schur) are preferred hosts, just like the other areas reported in other countries where OLBs presence has been confirmed [72,73]. OLB attack intensity between those two years does not differ significantly. In the area of origin, the species survives in less extreme conditions [72], which explains why the species has survived and thrived in the south-eastern area of Hungary from 2016 to 2017 [34,35] and in similar conditions in the western and southern areas of Romania [17]. In Sibiu, the 2021 minimum annual temperature was −0.84 • C and in 2022 (January-September) was −0.62 • C. Sibiu's climatic conditions now (in the last decade) are similar to the native area of origin, Eastern North America [17]. Climate Change would appear to have a very strong correlation to the establishment and now rapid growth of OLB in Sibiu and Europe. In the current context of the invasion in Sibiu, the most important step was to detect and monitor the species, to understand the behavior of this pest, to contribute to forming decisions based on scientific research on how best to control and limit the attack in the forest ecosystems in Sibiu County. During the two-year study in Sibiu, information on population abundance and environmental conditions in the area was documented, as well as the identification of factors influencing the development and reproduction of the invasive OLB populations [68]. Following our study, we conclude that the likely origin of the OLB infestation in Sibiu could be Cluj County, located 175 km from where the species was reported in 2017 in Baciu Forest [70]. The spread to Sibiu was possible via transportation, by wind and air currents.
As the specialists who have studied the ecology and biology of OLB agree, it can spread quickly via roads and railways in all stages (egg, nymphs and adults); also, the insect can be transported by pedestrians, car traffic and rail traffic [24,31,37,84,85].
The species can also be passively spread by the wind [24,37] and Sibiu has dominant winds (Mureşan wind that blows from Mures county), with high frequency and from a north-west direction, the mountain breeze and the so-called 'Great Wind' (snow eater), which manifests in early spring.
As soon as the presence of OLB was detected, we sought to rapidly identify all infected areas in Sibiu County. Early detection of infected host trees and applying control precautions reduced the chance of spreading to new areas. The published information until now about the spread of OLB in Romania [68] and other European countries can be helpful in predicting its likely future spread. In Sibiu, OLB was found mainly on oak species (Quercus spp.), but in the future, it can infest other types of trees such as elm (Ulmus spp.), lime (Tilia spp.) and rose (Rosa spp.) [37,[75][76][77][78][79][80][81][82][83][84][85][86]. In 2021-2022, damage to monitored trees (P1-P18) was caused by sap-feeding by adults and nymphs, causing a decrease in photosynthesis [42], premature defoliation and discoloration of leaves [37,61] and reduction of resistance to attack of other pests, diseases and sources of pollution [24]. Considering the adaptive capacity of OLB, we can conclude that this insect, which does not cause major damage in the native area, causes extremely severe damage in other geographical areas such as Sibiu. OLB can be considered a serious problem for both forest ecosystems and for trees in the city's parks and gardens, as it can affect the aesthetics of trees and, in the future, also the production of wood [67]. This conclusion is worrying in terms of the future of Quercus spp. Stands in Sibiu County, which in 2022 were 95% affected (P 1 ) by this invasion. We, therefore, intend to carry out more research in the future to determine what might prevent this species from spreading farther into the Sibiu region and find ways to eradicate it. We expect that in 2023, if climatic conditions are favorable, the increase and spread of attacks will extend throughout the county in all forest ecosystems because of the lack of natural limiting factors and lack of control methods in this stage of invasion. To limit the OLB attack in Sibiu County, we suggest that there is an urgent need to develop a set of biosecurity measures as well as to support research by the relevant bodies.