Environmental variables influence the developmental stages of the citrus leafminer, infestation level and mined leaves physiological response of Kinnow mandarin

Climate change has not only exacerbated abiotic stress, but has also rendered external conditions more feasible for pests to spread and infest citrus fruit. Citrus leafminer (Phyllocnistis citrella) is a potential pest that directly feeds the newly sprouted leaves and twigs of all three spring, summer and autumn flushes. Increasing temperatures in spring and autumn, leafminer accrued more heat units or developmental degree days to accelerate the biological stages of its life-cycle, thereby increasing the pressure of infestation. Present work was conducted at three different environmental conditions in Sargodha, Toba Tek Singh (TTS) and Vehari districts of the Punjab province, Pakistan; all three experimental sites were located in different agro-ecological zones. More infestation was recorded in all three flushes at TTS and Vehari than in Sargodha. Overall, more damage was observed due to higher temperatures in TTS and Vehari than in Sargodha. After May–June heat stress, spontaneous vegetative growth continued from July to November, produced newly spouted tender leaves for feeding the leafminer larvae, and was seen more in TTS and Vehari. Leafminer larva prefers to enter young and tender leaves with a maximum entrance in leaves up to 1 cm2 in size while observing no entrance above 3 cm2 of leaf size. Physiological response of leaves primarily attributed to chlorophyll and carotenoid contents, both of which were recorded lower in the mined leaves, thereby reducing leaf photosynthetic activity. Similarly, lower levels of polyphenols and antioxidant activity were also recorded in the mined leaves. The on-tree age of mined leaves of three vegetative flushes of Kinnow plant was also less counted than non-mined leaves. Climate change has affected vegetative phenology and become feasible for pests due to extemporaneous leaf growth, particularly leafminer, and eventually causes economic loss by supplying low carbohydrates either to hanging fruits or next-season crops.


Materials and methods
The present study was conducted in three major citrus growing regions of the province of Punjab, Pakistan, during the 2017-18 and 2018-19 seasons.
Weather data of three locations. Weather data from the respective experimental sites were taken from the Pakistan Meteorological Department and the average data for two years (2017 and 2018) is shown in Table 1.
Selection of orchards for data collection. The selected orchards were located at Sargodha (32 (30.0452° N, 72.3489° E and altitude 140 m) in Pakistan. Plants of identical features like age (12-15 years), healthy, vigorous with planting density (250-260 plants/ha) in block form having 2 hectares area were selected from three sites. Total 36 branches/twigs of lead pencil size were marked at different canopy positions of the plant 44 . Tagged branches represented three canopy positions (lower, middle and upper) from plant four directions and at each canopy position (inner, middle and outer) sides were used. Uniform cultural practices were followed in experimental sites. Data were recorded on a monthly basis during the growing season 2017-2018 and 2018-2019 31 .
Agrometeorological/Thermal indices calculation. Leaf miner threshold temperature and thermal constant. Threshold temperature of the leaf miner developmental stages (10.57, 7.31 and 7.42 °C) and thermal constant/developmental degree days (50.76, 109. 89 and 136.98 DDs) of eggs, larvae and pupae, respectively, were used in the calculation of agrometeorological/thermal indices 42 .
I-Developmental degree days (DDs). Threshold temperature of each developmental stage of leafminer (egg to pupa) was subtracted on a monthly basis from the mean daily temperature and expressed as °C day 28,30,45 .

II-Hydrothermal units (HYTUs).
Leafminer's individual developmental stages DDs were multiplied with each month's average relative humidity (RHa) for the calculation of HYTUs and expressed as °C day% 30,46,47 .

III-Photothermal index (PTI).
Individual stage developmental degree days (DDs) of leafminers were divided on the respective month time taken in days to complete the cycle of different phases and expressed in °C 30,48,49 .
IV-Helio thermal unit (HTU). Actual bright sunshine hours were multiplied by DDs and value articulated in °C day hours 30,44 .
Quantification of flushes and leafminer mined leaves. The number of leaves in each flush was calculated from the tagged branches and the percentage of flush was quantified by adding three flush leaves 50 . Similarly, in each flush, the mined leaves were also counted, and their percentage was calculated from the total number of mined leaves of each flush.
Monthly sprouting and leafminer larva entrance. Non-mined and mined leaves were calculated on a monthly basis from tagged branches, and the newly sprouted and leafminer larva entry leaves were also calculated. PP-Systems, CIRAS-3, Amesbury, U.S.A) was used to measure physiological response of non-mined and mined leaves. Net assimilation rate, stomatal conductance, sub-stomatal conductance/intercellular CO 2 concentration, transpiration rate and water use efficiency parameters were measured. Ambient sunlight and cuvette temperature with a standard reference of CO 2 (390 μmol mol −1 ) have been fixed (manual of the instrument available at www. ppsys tems. com) for measuring data of different mining lengths and intact leaves. The selected leaves were removed after taking photosynthetic activities to measure mining length. Mined area of the leaf was removed to measure percentage of damaged leaf. Data recorded on the PP-System/Infrared Gas Analyzer (IRGA) of different leaves with mining damage ranged from 10 to 60 per cent compared to non-mined leaves. Matured twomonth-old (mined and non-mined) leaves were used to estimate physiological response. Similarly, different age in months 1-8 of both non-mined and mined leaves physiological response was also checked through IRGA. Leaf cuvette dimension (18 × 25 mm) was set to cover an area of 4.5 cm 2 . Whole chamber was covered with leaf by inserting middle side of non-mined and infested portion of mined leaves to attain accuracy. Similarly identical features leaves free from insect-pests except LM of both non-mined and mined were used.

Results
Data analysis regarding leafminer damage and physiological response of mined leaves reflected significant influence under varying environmental conditions, which were explained and discussed under.

Egg (°C day %) Larva (°C day %) Pupa (°C day %) Egg (°C day %) Larva (°C day %) Pupa (°C day %) Egg (°C day %) Larva (°C day %) Pupa (°C day %)
Jan 3980. 89   Monthly sprouting and leafminer larva entrance. The perusal of data on monthly new sprouting leaves and damaged leaves from leafminer was shown in Fig. 2A,B, which showed significant impacts of different climatic conditions. Maximum newly sprouted leaves were recorded at Sargodha (41%) during March 2017 and minimum in TTS (3.33%) in the month of November 2018. In all three experimental sites, higher count of newly sprouting leaves were seen in March and lower during July and November. Similarly higher LM infestation was seen in March and lower in February and November in all three sites. However, maximum mined leaves were counted at Vehari (8.66%) in March 2018 and lower in Sargodha (0.25%) in the month of July 2017. In all three districts, no new sprouting was seen in January, April, May, June and December and henceforth, LM infestation was not recorded as such in these months, as larva just after egg-hatching prefer to feed or make zigzag entry in newly emerging leaves.
Leaf size entrance by larva after egg-hatching. Leaf size mined by leaf miner was presented in Fig. 3.
Maximum larva entry/mining was recorded in leaf size (0-1 cm 2 ), followed by leaf size (1-2 cm 2 ) and least in leaf size (2-3 cm 2 ) at all three districts. In leaf size (0-1cm 2 ), maximum larva entry was found (63.33%) in spring flush and minimum (53.33%) in autumn flush at Sargodha. In leaf size (1-2cm 2 ), higher larva mining was recorded at Sargodha (37.33%) in autumn flush and lower at Vehari (30.67%) in spring flush. However, least mining was recorded in leaf size (2-3cm 2 ) by recording more in autumn flush at Vehari (9.67%) and less in summer flush at Sargodha (4.33%). Leaf size above 3 cm 2 was shown to be tolerant of newly hatched larva making mine or leaf entry in all three locations. In all three districts and flushes a significant differences were observed with respect to leaf size in the larva making mine or entry.

Sites & vegetative flushes
Larva entrance (%) Figure 3. Larva entrance after egg-hatching of leaf size. Bars sharing dissimilar letters are significantly differed according to LSD test (P ≤ 0.05).Leaf size in cm 2 .   Table 12 and trend pattern in Fig. 5A,B. Physiological activity has shown a higher response in non-mined leaves than in mined leaves (10-60%) of damage levels. Maximum net assimilation rate (4.3 μmol CO 2 m −2 s −1 ), stomatal conductance (58 mmol H 2 O m −2 s −1 ), sub-stomatal conductance (246.33 μmol mol −1 ) and water use efficiency (4.17 mmol CO2 mol −1 H 2 O) were recorded in non-mined leaves. In contrast to other physiological activity, there was a lower rate of transpiration in non-mined leaves (1.03 mmol H 2 O m −2 s −1 ) than in mined leaves (10-60%) of damage. www.nature.com/scientificreports/ Photosynthetic activity of non-mined and mined leaf at different age. Photosynthetic activities of non-mined and mined leaves are shown in Fig. 6A-C.
Maximum photosynthetic activities such as net assimilation rate, stomatal and sub-stomatal conductance were recorded in non-mined leaves and minimum in mined leaves. Increased trends in carbon assimilation, stomata and sub-stomata conductance/intercellular CO 2 concentration were observed in old leaves (1-5 months) and then slowed down and began to decline in both non-mined and mined leaves. Maximum carbon assimilation    Leaf age of non-mined and mined leaves. The leaf-age of non-mined and mined leaves was shown in   Plant phenological growth trend in fluctuating weather conditions. Phenological growth trend is given in Fig. 7 and based on field based study.

Discussions
Agrometeorological/Thermal indices. Temperature was recorded more at Vehari and average relatively humidity (RHa) at Sargodha 2 , henceforth more DDs, PTI and HTU were available at Vehari. Similarly, in Sargodha, DDs were counted less than in Vehari, but vice versa in the case of RHa; as a result, both districts had less HYTUs counts than TTS. Average relative humidity (RHa) was seen more at TTS than Vehari and mean daily temperatures were observed higher than Sargodha, henceforth more HYTUs were available at TTS on a monthly basis at different leafminer developmental stages. Bevington and Castle 32 reported that agrometeorological indices were fluctuated location-wise due to climatic factors variation in changing seasons. Similar results for additional growing degree days (GDDs) of crops have been reported in warm regions 33,34 . In the present work, mean daily temperature was recorded more at Vehari, followed by TTS and lower at Sargodha, therefore DDs were calculated by keeping different developmental stage threshold temperatures. The calculation of other agrometeorological indices were based on DDs and climate variables that also influenced the developmental stages of insect-pests 35 . Just as plant growth phases are directly linked to agrometrological indices, as in the case of citrus fruit growth phases 44 , the developmental stages of leafminer are also affected by climate variables 38 . DDs availability can determine the span of life cycle of leafminer and the number of generations all year round 42 . In addition, leafminer population pressure on citrus in a specific area is directly linked to the availability of agrometeorological indices 40 and emerging flush 25 . More climate variations have been observed in citrus-growing three sites, and henceforth fluctuating agrometeorological indices have been computed 31 , while global warming has increased temperatures 20 and biotic stress 19 . As a result, more DDs and other agrometeorological indices were available to leafminer at different developmental stages. More infestation was observed in TTS and Vehari in the present work to indicate that the leafminer egg to pupa stages took less time with more generations in a year. The developmental stages of leafminer (egg, larva and pupa) squeeze at high temperatures 25,43 . Nevertheless, different developmental process of leafminer ceases below threshold temperature 42 . Singh 51 also reports that leafminer has shown more growth and rapidly completed egg to pupa stage at high temperatures and prolongs developmental process in cool months while studying Kinnow and rough lemon plants. In current work, more DDs, PTI, HTU were computed in warm districts and summer months, so more pest infestation was observed www.nature.com/scientificreports/ in summer flush at TTS and Vehari. Pinto and Fucarino 36 and Santos et al. 37 report rapid developmental rates of different stages of leafminer (egg to pupa) in high photoperiod areas, while more HTU and PTI were available in warm regions and summer months in the current work to demonstrate that rising temperatures not only increased DDs but also increased agrometeorological indices in changing climate scenarios. Weather conditions decide on the available monthly basis agrometeorological indices 45 to indicate the number of generations of leafminer throughout the year 41 and the seasonal life cycle in a given area 39,40 . Fluctuating agrometeorological indices were computed in Kinnow growing three districts in climate change scenario, which has changed plant growth patterns in summer flush and also hastened the developmental process of leafminer as this work is justified by population model of insect pests based on meteorological factors and availability of resources 38 .
Kinnow flushes and leaf miner damage. Newly sprouted leaves have thin epidermis and leafminer attacks are seen higher 60 . Similar results were observed in all three flushes in this study. In Kinnow mandarin, spring flush was counted 55-60%, followed by summer 25-30% and autumn 10-15% 50 which justified current work on flushes. The highest oviposition rate of leafminer was recorded at 30 °C 25 while similar temperatures were observed in the three districts during March. Relatively higher temperatures were recorded at Vehari and TTS during the last week of February to the end of March; more leafminer infestation was counted on newly sprouted spring flush leaves as this pest had overcome winter hibernation earlier. At high temperatures, more agrometeorological indices were available to leafminer and can quickly complete life cycle while squeezing the developmental stages (egg to pupa). Abo-Kaf et al. 61 report leafminer oviposition period 2.28 days at 30 °C which changes its duration in the changing temperature regime 25 . In current work, more agrometeorological indices were available to leafminer during the summer months, which produced overlapping generations. As a result, more larva mining was observed at TTS and Vehari, because in summer flush more growing degree days were accumulated to produce spontaneous vegetative growth. In autumn flush, less damage was seen due to low temperatures and no oviposition of the leafminer occurred at or below 15 °C 25 . Egg, larva and pupal developmental cycles were shortened with rising temperatures 25 . In warm conditions and summer months, more agrometeorological indices were available, while leafminer accelerated the life cycle by producing overlapping generations. As a result, more infestation was recorded in warm districts of Vehari and TTS than in Sargodha. Similarly, less agrometeorological indices were available in Sargodha in autumn and spring flushes, therefore less pest infestation was observed. In addition, heavy rainfall also slowed the growth of leafminer in the summer months, and more rainfall was reported in Sargodha, resulting in less infestation in the summer flush.
Monthly sprouting and leaf miner larva entrance. Due to the winter hibernation of the leafminer, less infested leaves were reported on a monthly basis in February, but a sudden rise in temperature during March coincided with heavy spring flush, resulting in higher damage in all three districts as the optimal oviposition temperature available 25 . More agrometeorological indices were available in the warm districts of TTS and Vehari, with an increase in the pest population due to the rapid rate of developmental stages resulting in more infestations. High temperature in July to October optimized conditions for leafminer by recording more infestation in warm districts due to additional accretion of DDs, PTI and HTU. In the same way, the increase in the growing degree days (GDDs) in warm months led to spontaneous slow vegetative growth. As a result, newly sprouted leaves ensnared adult female for oviposition and hastily completed egg-hatching stage with more first instar larva population to feed tender young leaves. No vegetative growth occurred in the months of January, April, May, June and December, and henceforth no infestation was seen on mature leaves as the female preferred young leaves to lay eggs 62 . These findings are substantiated by the work of 63 who reported that the peak mining period was February-March and July-October. Rainfall in Sargodha was higher than TTS and Vehari during the spring, summer and winter periods and has adverse effects on leafminer. Similar adverse effects of rainfall on the developmental stages of leafminers are observed during monsoon in Bangladesh 64 justifying this study.
Leaf size entrance by larva after egg-hatching. Mostly, the leafminer prefers young and tender leaves 8 of a size (10-25 mm long) for oviposition 62 . The first instar larva hatched from the eggs immediately feeds on tender epidermis tissues and begins to mine zigzag in newly sprouted leaves 10 while the first instar larva to the pupal stage continues to mine leaves to feed on spongy parenchymatous cells 60 . Maximum 60-63 percent of the larva entry was seen on a leaf size of up to 1 cm 2 , while 30 percent larva mining in the leaf size (1-2 cm 2 ) was observed symmetrically in three districts. The larva making leaf size (2-3 cm 2 ) mine was recorded to be the lowest in all districts (4-8%). Leaf size larger than 3 cm 2 reached hardness for the entry of larvae or mine formation, suggesting that the small leaves (1-5 days) were more infested than the larger leaves. Leaf aged 11-15 days is resistant to entry/mining of larvae and leafminer population pressure on citrus is influenced by the availability of young leaves and weather factors 51 . Similar trends in newly emerging leaves have been observed in the present work, indicating that the larva preferred young, tender and emerging sprouts of all three flushes. Vercher et al. 65 also observed that the first instar larva feeds on young leaves (10-20 mm long) justifying extra attacks on small leaves in the present work.
Leaf sclerophylly for leaf miner larva feeding after entrance. Female leafminer tends to lay eggs on emerging tender leaves 62 and larvae enter epidermis soft tissues 10 and make zigzags to feed on parenchymal spongy cells 60 . Among citrus leaf sclerophylly parameters, succulence determines leaf tenderness 50  www.nature.com/scientificreports/ the inner tolerance of the leaf against the entry of the larva was increased earlier in the spring and summer, and later in the autumn. The life cycle of leafminer depends on the availability of young leaves and external weather conditions 51 . In the spring and summer seasons, leafminer larva finished fast feeding while it extended feeding in the autumn due to tender tissue availability, in addition to being dependent on prevailing environmental conditions. In spring and summer flushes when more DDs were available, a rapid larva feeding was observed. While less DDs and other agrometeorological indices were calculated in late autumn and more leaf succulence was also recorded, which provided more space for larvae to feed until favorable external conditions were established for the next life cycle.
Chlorophyll and carotenoids contents of non-mined and mined leaves. Chlorophylls and carotenoids pigments are essential in plants. Chlorophylls are actively involved in photosynthetic activities 66 , and channel solar radiant to assimilate atmospheric CO 2 to organic carbon compounds 67 . However, carotenoids transmitted light for photosynthesis 68,69 as well as protected leaves against harmful effects of solar radiation 70,71 by stabilizing proteins in the photosystem 72 . In mined leaves, total chlorophyll, chlorophyll a and b, and carotenoid content were reduced due to feeding of mesophyll tissues and chloroplast depletion in leaves. Chen et al. 15 reported similar findings from mined leaves in a mangrove plant (Avicennia marina). Chlorophylls and carotenoids are directly involved in photosynthetic activities 73 and ascertain leaf age on the tree while their reduction in mined leaves has slowed photosynthetic activity and reduced leaf age by inducing the cycle of leaf abscission 74 .
In this study, chlorophylls and carotenoids were found to be lower in the mined leaves in all three districts and in the three vegetative flushes.

Polyphenols and anti-oxidant activities of non-mined and mined leaves. Reactive oxygen and
nitrogen species (ROS/RNS) are essential for chemical signaling, energy supply and defense mechanisms, but their overproduction under stress conditions or exposure to external oxidizing processes has caused failure of defense mechanisms and damage to key biochemicals such as DNA, protein, lipids 75 . Antioxidants can prevent oxidative damage 76 and polyphenols have a positive antioxidant correlation 77 , while phenolic acids, flavonoids and flavonols are the main sources of citrus antioxidants 78 . Flavonoids have protein binding function while flavonoids bind proteins to cellular receptors and transporters 75 . Chlorophylls also work as an antioxidant compound in leaves 79 which has been shown to be lower in mined leaves than non-mined leaves, and polyphenols such as total phenolic, flavonoid and flavonoid contents have a positive correlation with antioxidant activity, therefore less reported in leafminer infected leaves. Similarly, polyphenols have also been found to be lower in mined leaves due to loss of vital tissues and chloroplasts, indicating that mined leaves have less polyphenol and antioxidant activity than non-mined leaves.
Physiological responses of non-mined and mined leaves. Non-mined leaf photosynthetic rates are higher than herbivores damaged leaves 17,80 , but leaf physiology is influenced by degree and damaged tissue types and C-compound relocation from primary to secondary metabolism 81 . A lower rate of net assimilation, stomatal conductance and water use efficiency were recorded in mined leaves than non-mined leaves. Similarly, leaf miner larvae feed parenchymatous tissues to varying degrees by affecting the physiological response of the leaf to the level of the mined/damaged leaves. As the damage to the leaves increased, photosynthetic activities have been more affected 82 . Similar trends have been recorded in mined leaves (10-60%) with a gradual decline in photosynthetic activity. Transpiration rate remains high in damaged leaves due to loss of epidermal and cuticle layers 83 which also justified high transpiration in the Kinnow mandarin mined leaves in current work. Stomatal conductance recorded less in mined leaves that assimilated low carbon and also lowered photosynthetically water use efficiency 80 while damage or loss of cuticle layer increased transpiration 83 . Similar findings in the current research have been recorded for damaged Kinnow mandarin leaves. Mined leaves have lost chloroplasts and degenerated thylakoids, which have reduced photosynthetic efficiency. Increased extent of mined leaves directly lowered net assimilation, stomatal and sub-stomatal conductance while accelerating transpiration rate. As a result, water use efficiency of leaf was declined. A similar trend was seen in this study of different levels of damage (10-60%). The present findings are consistent with the work of 18 on Pastinaca sativa L. In contrast to nonmined leaves, decreases in stomatal activity in mined leaves 84 have not lowered transpiration rates due to loss of cuticle layer 83 . As a rule, low carbon is assimilated and more water loss in transpiration, thus photosynthetically reducing the water efficiency of the leafminer infested leaves. Sub-stomatal conductance is actually inter-cellular CO 2 concentration which has shown to be lowered in mined leaves due to damage of mesophyll and thylakoids tissues. As a result, low level of photosynthetic efficiency of the leaf was observed in damaged leaves of Kinnow mandarin. Similar findings are observed in the photosynthetic and gaseous exchange rates of citrus by recording a low physiological response of larval feeding leaves 80 .
Photosynthetic activity of non-mined and mined leaf at different age. Photosynthetic activity of mined leaves remains lower 80,85 due to feeding of parenchyma tissues 86 that alter physiological performance of the leaf 85 . Significant reductions in net assimilation rate, stomatal and sub-stomatal conductance were observed in mined (1-8 months) old leaves compared to non-mined/intact leaves in the present work. Costa et al. 16 also reported low photosynthetic activity in leafminer damaged melon plants, which also confirmed low photosynthetic activity in mined leaves as recorded in this study. Dented chloroplasts in mined leaves reduce carbon assimilation 80 and respiration rate 87 due to low carbon accumulation or CO 2 emissions in spongy parenchymal cells 88 which reduce sub-stomatal conductance and ultimately delay the physiological response of damaged leaves 89 . In the same way, stomata below the mined area also responds to closure or opening but is impaired in function due to malfunctioning of the stomatal aperture that has decreased stomatal conductance 85  www.nature.com/scientificreports/ recording of low net assimilation rate, stomatal and sub-stomatal conductance compared to non-mined leaves 1-8 months old. More reduction in photosynthetic activities was observed in mined leaves after the 5th month due to the prior start of senescence cycle. The present findings of low photosynthetic activity of the mined leaves are consistent with the work of 80,90 on citrus 91 , on tomatoes and 92 on Milkweed (Asclepias syriaca).
Leaf age of non-mined and mined leaves. Leafminer damage young and tender leaves by mining on the epidermis tissues 10 . Leafminer feeds on parenchymatous cells 60 and destroys mesophyll and thylakoid tissues as well as the cuticle layer 83 and eventually deprives the leaves of vital components such as chlorophylls, carotenoids and polyphenols by reducing antioxidant activity 15 . In addition, mined leaves have low photosynthetic activity and incapacitated physiological response 85 . As a result, the leaf senescence cycle is accelerated as observed in current research. Mined leaves age was reported to be low than non-mined in all three Kinnow mandarin vegetative flushes. Mined leaves have a low physiological response 93 and a weak defense mechanism against weather vagaries 94 . As a result, leafminer infested leaves reduced physiological efficiency and also reduced on-tree age by causing economic loss 95 of less carbohydrate supply 80 to the rest of the plant due to impaired xylem and phloem function 96,97 . In mined leaves, the abscission rate is more than intact leaves 74,98 , while infested leaves have active abscission 99 . The same trend of abscission was seen in this work of recording the earlier shedding of the mined leaves in all three flushes.
Plant phenological growth trend in fluctuating weather conditions. Kinnow plant phenological growth trend in fluctuating weather conditions has favored more infestation of pests, especially leafminers. Floral and vegetative growth simultaneously begins in the spring season and maximum leaf growth and tender twigs are recorded about 60 per cent in spring flush 1 . Global warming has risen temperatures over the last century 20 by inducing abiotic and biotic stress on crops 19 and also on citrus 31 . In citrus growing areas, the autumn and spring seasons were squeezed 2 while more agrometeorological indices were recorded under warm conditions and also during the summer months 44 . As a result, more growing degree days (GDDs) to plant and developmental degree days (DDs) to insect pests were available by altering the plant vegetative growth pattern and accelerating developmental stages of insect pests. Spring flush began maximum photosynthetic activity from fruit cell enlargement until the on-tree hanging fruits arrived at maturity. The second contribution of the net carbon assimilation is the summer flush, which provides energy to the hanging fruit in ripening stage as well as the newly sprouted floral and vegetative growth during the spring season. Autumn flush leaves assimilate less carbon at an early stage, which reached its peak during fruit-setting until the cessation of cell division. Leafminer damaged leaves of three vegetative flushes provide less carbohydrate due to low carbon assimilation in either hanging fruit or next-season crops. Fluctuating weather conditions have resulted in uneven growth patterns of leaves that favor overlapping leafminer generation to rapidly proliferate due to availability of more DDs. Fruit-set in the spring season, requiring maximum energy at cell division and cell enlargement phases, is likely to continue throughout the summer season. The autumn flush leaves assimilate maximum carbon by supplying energy to fruit during cell division, while the spring flush leaves are the main source of plant energy with an overwhelming source during the fruit cell enlargement phase and have also contribution in the maturing phase. Citrus leaf attained a maximum level of chlorophylls and carotenoids at the age of two and a half months, as the results begin with peak carbon assimilation, which declined at the senescence stage about 1 month before the leaves were shed. Mined leaves begin to shed ahead of time when other parts of the plant require more carbohydrate by causing direct economic loss of less energy supply to fruit 80 . In climate change scenario, global warming caused extreme heat-wave at the start of summer season 2 that has retarded net assimilation of carbon with earlier drop of summer flush leaves along with burning of tender leaves of spring flush. As a result, low carbon assimilation resulted in more fruit drops and warm-humid conditions in late summer, resulting in more carbon assimilation to spontaneously produce new sprouts with higher pest pressure, particularly leafminer. In the same way, both temperature and humidity increased during the autumn season, resulting in more carbon assimilation while inducing new sprouts to lure more pests until the beginning of winter. More leafminer pressure was observed in the current work at warm districts. Erratic weather behavior has adversely affected pest-damaging leaves in the climate change scenario; especially leafminer infested leaves. Climate change, on the one hand, has negatively affected the physiology of citrus plants; on the other hand, it has provided favorable conditions for the spread of pests, particularly leafminer.

Conclusion
Leafminer has emerged as a serious citrus pest in a climate change scenario. More infestation was observed in the high-temperature TTS and Vehari districts during all three vegetative flushes, as more agrometeorological indices were available to accelerate the life cycle of the leafminer. More leafminer damage was recorded in the leaf size (0-1 cm 2 ) while 12-15 days old leaves above 3 cm 2 were tolerant. Spontaneous leaves grew more in TTS and Vehari between July and November than in Sargodha, with more leaves being mined. Mined leaves contained less chlorophylls, carotenoids and polyphenols with low antioxidant activity than non-mined leaves. The physiological response of the mined leaves remained low, affecting fruit yield and quality. These findings would be useful in the future to develop strategies by knowing the impact of leafminer on citrus in a changing climate scenario. Based on climate variables, the availability of agrometeorological indices will determine the size of the leafminer population and the number of generations over the year, as well as the growth pattern of the citrus plant vegetative flush.