НАУКА ЗА ГОРАТА, КН. 1, 2020 FOREST SCIENCE, No 1, 2020

VARIABILITY IN THE PHENOLOGY AND ECOLOGY OF the PINE PROCESSIONARY MOTH (THAUMETOPOEA PITYOCAMPA) IN THE SOUTH-EASTERN RHODOPES,

Gergana Zaemdzhikova, Georgi Georgiev, Margarita Georgieva, Maria Matova, Plamen Mirchev Forest Research Institute – , Bulgarian Academy of Sciences

Abstract: Studies on the phenology and ecology of the pine processionary moth (Thaumetopoea pityocampa) were carried out in plantations of the black pine (Pinus nigra) in four localities in the South-Eastern Rhodope Mts. – near the town of Ivaylovgrad and in the region of Kirkovo, the Kandilka and Austa Villages. A phenologically atypical popu- lation “summer form” (SF) was established on the territory of the Kirkovo and Kandilka Villages. A population following the typical cycle, a “winter form” (WF), was observed in the region of Ivaylovgrad. Both phenological forms of T. pityocampa co-existed around the Austa Village. There 20% of the studied nests were empty, likely owing to the fact that the caterpillars had gone down into the soil for hibernation; in the rest of the nests – overwin- tering caterpillars were established. In two localities with different forms of pine processionary moth – Ivaylovgrad and Kirkovo, the egg parasitoid complex was explored. No significant differences were found between the parasitoid-host relationships. The dominant parasitoid in both habitats was Baryscapus servadeii, followed by the polyphage Ooencyrtus pityocampae. In all of the locali- ties, the hyperparasitoid Baryscapus transversalis presented infesting an average of 5% of established parasitoids.

Key words: Thaumetopoea pityocampa, phenology, ecology, Bulgaria

INTRODUCTION

The pine processionary moth Thaumetopoea pityocampa (Denis & Schiffermüller, 1775) (Lepidoptera: Notodontidae) is a Mediterranean species, whose larvae are the main defoliator of pine trees. Its harmfulness for pine forests is multidimensional: besides the economic losses, e.g. decrease of tree growth through and dieback of the attacked forests, the hairy caterpillars are a dangerous allergen to humans and animals and threaten the development of sea and mountain resorts. Throughout the world range of the pine processionary moth, in particular in Bulgaria, an expansion of the species occurrence has been observed in recent years (Mirchev et al., 2011b). The factors determining this process can be sought both in climate change and afforestation of new pine forests, as well as in some features of the species’ biology and phenology.

101 The limiting factors for the survival of the eggs and caterpillars of the pine processionary moth are high summer and low winter temperatures, as well as the absence of “favourable days” in winter with temperature suitable for feeding and development of the caterpillars (Battisti et al., 2005). There are two phenological forms of the pine processionary moth, “summer form” (SF) and “winter form” (WF), as it has been already accepted in the entomological literature (Battisti et al., 2015). Аt the typical Mediterranean form (WF), the larvae overwinter in the nests and are characterised by the winter feeding. Its pupation takes place at the end of the winter in the soil. Тhe larval development stage of the SF is completed at the beginning of December and the caterpillars leave the nests before the beginning of the winter and burry into the soil to hibernate. The summer development of eggs and young caterpillars, which has its disadvantages and risks for the species, is obviously an adaptation of the species to the continental climate. It allows the species to avoid overwintering in tree nests, which is deadly in a harsh winter. The summer form of Т. pityocampa has been reported for the first time in Bulgaria in 1950s in the sub-Balkan valleys and the central part of the Rhodope Mts. The development of its larvae ends before the winter cold months (Tsankov, 1960). In a more recent study, a phenologically atypical form with early adult emergence in summer has been found in Portugal, in the southern part of the Lieiria the National Pine Forest, which co-exists with a population with typical winter larval development in the same area (Santos et al., 2007). Santos et al. (2013) reported differences in biology and morphology of both forms. In recent years, an expansion of the range of the pine processionary moth’s summer form has been established in the Eastern Rhodope Mts. in Bulgaria (Mirchev et al., 2017; Zaemdzhikova et al., 2018; Georgieva et al., 2019; Mirchev et al., 2019). The aim of the present study was to explore the phenology of Т. pityocampa in the South-Eastern (SE) Rhodope Mts., as well as to compare the impact of dominant egg parasitoids on the two phenologically different forms.

МATERIAL AND METHODS The experimental material was collected at four localities in the SE Rhodope Mts.: near the town of Ivaylovgrad (protected area “Dupkata”), Kirkovo Village (the area “Krastavo Bradtse”), Kandilka and Austa Villages (Figure 1). The distance between the two most distant objects Ivaylovgrad and Kirkovo is 64 km by airline. All localities fall into the Continental-Mediterranean climate region, characterised by mild and very humid winter and hot and dry summer. Unlike the habitats of the pine processionary moth in south-western

102 Fig. 1. Studied localities of Thaumetopoea pityocampa in Bulgaria

Bulgaria, the SE Rhodope Mts. are considerably less protected from cold air. In physical-geographical aspect, the characteristics of the four sites are similar. They are located in the Bulgarian part of the SE Rhodopes, which have a low mountainous and hilly relief. The host plant in all localities is Pinus nigra (Arn.) and the altitude is about 500 m a.s.l.. Only the northernmost site, the protected area “Dupkata” near Ivaylovgrad, is at a lower altitude (Table 1). In each locality, during the period December-February (2010-2019), the pine processionary moth’s nests were observed by dissection, to determine the proportion of nests that were left by the caterpillars to pupate and overwinter in the soil. At the end of the caterpillars’ hatching period, egg batches were collected and placed individually in tubes. In laboratory conditions, they were scaled and the eggs with parasitoid’s emergence were marked. The final analysis was performed after the parasitoid emergence had been over. The parasitoids emerging before the collection of egg batches were determined by their meconia and remains as reported by Schmidt, Kit (1994), Tanzen, Schmidt (1995), Schmidt et al. (1997), Tsankov et al. (1996; 1998). Egg parasitoids were divided based on their relative abundance (RA) into two groups, according to the Skufin criteria (1949): dominant species – the number is over 8% of all emerged parasitoids; subdominant species – the number is between 2 and 8% of all emerged parasitoids.

103 Table 1. Main characteristics of the localities

Elevation, m Orientation Locality Latitude Longitude Host tree a.s.l. of the slope Ivaylovgrad 250 41°31’40” 26°07’07” E Kirkovo 459 41°37’82” 25°32’16” W Pinus nigra Kandilka 465 41°40’94” 25°58’18” N Austa 535 41°28’19” 25°30’35” E

RESULTS Phenological results The localities on the territories of Kirkovo and Kandilka Villages were occupied by the SF, while in the protected area “Dupkata” near Ivaylovgrad the plantations were inhabited exclusively by the WF. In a study in February 2017, a pine plantation near Austa Village was inhabited by both forms. It was established that nearly one-fifth of the nests were left by the caterpillars (belonged to SF), while in the other nests, the caterpillars was found to overwinter in them (belonged to WF; Table 2). Ecological results In two sites inhabited by the separate phenological forms – Ivaylovgrad and Kirkovo, an aspect of the ecology of the pine processionary moth was studied – the structure-determining egg parasitoids. Both habitats have similar physiographic characteristics and climate conditions in the summer and early autumn (when the females of the pine processionary moth lay eggs), excluding the difference in altitudes. As far as parasitoids were concerned, the important difference was that in the SF’s habitat near Kirkovo, the eggs of the host and parasitoids emerged in June and August when the highest temperatures during the year were recorded. On the other hand, in the WF’s habitat near Ivaylovgrad, the development of eggs and parasitoids occurred a month or two later, at moderate temperatures. In spite of this significant ecological difference, we recorded mostly similarities between the two localities in term of the parasitoid-host relationships. The dominant species Baryscapus servadeii (Domenichini, 1965) (Hymenoptera: Eulophidae) was the most numerous, followed by Ooencyrtus pityocampae (Mercet, 1921) (Hymenoptera: Encyrtidae) – a polyphage, the share of which varied widely. In the sample taken on 15.09.2016 in Ivaylovgrad, B. servadeii was below 8%. At the same site, in three out of four samples, the polyphagous Anastatus bifasciatus (Geoffroy, 1785) (Hymenoptera: Eupelmidae) was found with a share of about 10%, while in Kirkovo – it was either absent or rare. Adaptability to the large temperature

104 Table 2. Percentage of T. pityocampa colonies hibernating in soil

Number of Colonies hibernating Locality Date studied nests in the soil, % 2011, January 20 10 2015, January 20 10 Ivaylovgrad 0.0 2017, February 23 9 2017, December 14 5

2017, December 14 20 Kirkovo 100.0 2019, December 07 20 Kandilka 2019, December 07 20 100.0 Austa 2017, February 23 11 18.2 differences during its development showed also the hyperparasitoid Baryscapus transversalis Graham, 1991 (Hymenoptera: Eulophidae). It was present in all samples but was more numerous in the pine processionary moth’s habitat in Ivaylovgrad as compared to the one in Kirkovo (Table 3).

DISCUSSION The results of the present study showed that in a relatively small area, such as the SE Rhodope Mts., there were clearly different phenological forms of the pine processionary moth that co-existed on the same area in some localities. The climate factors could not be attributed the leading role in the formation of these two phenological forms. The importance of climate for the species composition of egg parasitoids and their relative share should also be revised. Studies of the phenological forms of the pine processionary moth in Bulgaria date back to the middle of the 20th century. A study in 1956 found that the caterpillars in the region of the , and Towns in the south-western Bulgaria hatched in August, overwintered in their nests on the pine tree, and pupated in the soil between April and June. In the central southern part of the country, however, (close to the and Hissar Towns and Village), caterpillars hatched from the eggs in late June and early August. In mid-October, about 40% of the caterpillars went down into the soil (Tsankov, 1960). Recent studies in the Eastern Rhodope Mts. have reported as “phenologically clean” habitats of the SF in the vicinity of the Kirkovo, Dobrostan (Zaemdzhikova et al., 2018) and Kandilka Villages (Mirchev et al., 2019). In Kirkovo, the hatching period of caterpillars lasted about one month: from the beginning of July to the first weeks of August (Georgieva

105 Table 3. Dominant and subdominant egg parasitoids of T. pityocampa

Total Total Subdominant para- Dominant parasitoids Lo- Date of number number sitoids cality collection of egg of para- batches sitoids Name RA* Name RA B. servadeii 62.4 - - O. pityocampae 29.0 - - 27.03.2010 22 1077 A. bifasciatus 10.6 - - B. transversalis 8.9 - - B. servadeii 48.4 B. transversalis 7.4 17.11.2010 31 1982 O. pityocampae 35.1 - - A. bifasciatus 9.0 - - I vaylovgrad B. servadeii 45.9 B. transversalis 4.5 27.03.2010 32 1982 O. pityocampae 38.4 - - A. bifasciatus 10.8 - - B. servadeii 85.6 O. pityocampae 7.2 15.09.2016 39 2487 - - A. bifasciatus 5.7 B. servadeii 62.4 A. bifasciatus 5.9 26.09.2016 137 4792 O. pityocampae 29.0 B. transversalis 2.5 B. servadeii 59.6 B. transversalis 5.0 Kirkovo 18.09.2018 197 6675 O. pityocampae 35.2 - - *RA – relative abundance et al., 2019). For comparison, the hatching period in the Sandanski region, occupied entirely by the WF, began in the middle of September (Mirchev et al., 2017). The present study shows that at short distances there are habitats of two different phenological forms. The distance between Kandilka and Austa Villages is 9 km by air. Similar observations have also been made elsewhere in the Rhodope Mts. Near – in the Dobrostan Village, only the SF of T. pityocampa was established, while ten kilometres away from it (close to the Muldava Village) – only the WF was found (Zaemdzhikova et al., 2018). The distances Austa-Kandilka and Dobrostan-Muldava are significantly smaller than the flight range of the male butterflies of the pine processionary moth (Mirchev et al., 2013). An even earlier development of the pine processionary moth has been found in Portugal. Тhe larval development stage of the SF, discovered in 1997, was between mid-June and September. This form overwintered as a

106 pupa in the soil, while the moths’s period of emergence and the timing of oviposition began in May (Santos et al., 2007). The same area is inhabited also by the typical WF but the ontogenetic development stages of both forms (caterpillars and adults) do not coincide in time. The males of the SF start flying in early May with a peak in early June, while the males of the WF start flying three months later and finish in early October with a peak at the end of August (Santos et al., 2007). This is considered as the first steps of a sympatric differentiation that could ultimately lead to sympatric, allochronic speciation. However, a study by Santos et al. (2011) at the same site in Portugal has proven some hybridisation between the two forms. Santos et al. (2013) also found morphological differences between the two forms in Leiria: the size of the eggs and the shape and colour of the scales covering the egg batches were different. They also report differences in the ecology of the SF, which are characterised by lower levels of egg infestation with parasitoids. There are also differences in the egg parasitoids’ complex. Unlike our study, in which B. servadeii was the dominant parasitoid for both forms, in Portugal, 95.2% of the number of parasitoids established in the summer form’s eggs belonged to O. pityocampae and Trichogramma embryophagum (Hartig), with a similar share of host’s parasitised eggs. One of the leading factors that could form different levels of survival in both forms and the parasitoids that are associated with them is the air temperature in certain periods of their ontogenetic development. Huchon and Démolin (1970) report that temperatures above 32°C could be extremely dangerous for eggs and young caterpillars. Masuti (1964) in his research has found that O. pityocampae is susceptible to high temperatures and habitats with prolonged periods of temperatures above 30° C are unsuitable for it. Furthermore, its larvae are quite vulnerable to changing the natural daytime rhythm of temperature. In contrast, the species of family Eulophidae, including B. servadeii, are more plastic and develop successfully even at higher temperatures. It should be noted that O. pityocampae is a polyphage. Its abundance depends on the presence and the abundance of its wide range of hosts. The comparison between the two summer forms of the pine processionary moth – in Bulgaria and Portugal, shows significant differences in their phenology and ecology. From a phenological point of view, the summer form in Portugal is more distinguished from the WF, since in Bulgaria, the periods of development of caterpillars, eggs and adults for both phenological forms are not completely different. In Portugal, the male adults’ flight of two forms runs at different times, while in Bulgaria, to some extent, there is a period overlaps. From an evolutionary point of view, of which the principle is the adaptation of the species to the conditions, there is no reason to believe that in Bulgaria there will be shifts in the phenology of the SF approaching these

107 in Portugal, unless changes occur in the climate or with further expansion of the species to the north. According to official information (EFA, 2010-2019), outbreaks requiring aerial pest control have been recorded in the region of Kirkovo every 2-3 years during the last decade. The differences in the ecology of the summer forms of T. pityocampa from Bulgaria and Portugal are also significant, in particular in their egg parasitoids, which could be attributed to the marked differences in the timing of the instar development. The much closer time periods of the appearance of the eggs of the two forms in Bulgaria are a serious reason for explaining the high number of the specific egg parasitoid B. servadeii.

Acknowledgements: This study was supported through the project “Expansion of pine processionary moth (Thaumetopoea pityocampa (Denis et Schiffermüller, 1775) (Lepidoptera: Notodontidae, Thaumetopoeinae) in Bulgaria – a dangerous allergen and economically important pest in the pine ecosystems” funded by the National Scientific Fund (DN01/17, 22.12.2016).

References

Battisti, A., M. Stastny, S. Netherer, C. Robinet, A. Schopf, A. Roques, S. Larsson. 2005. Ex- pansion of geographic range in the pine processionary moth caused by increased winter temperatures. – Ecological applications, 15 (6), 2084-2096. Battisti, A., M. Avcı, D. N. Avtzis, M. L. Ben Jamaa, L. Berardi, W. Berretima, M. Branco, G. Chakali, M. A. El Alaoui El Fels, B. Frérot, J. A. Hódar, I. Ionescu-Mălăncuş, K. İpekdal, S. Larsson, T. Manole, Z. Mendel, N. Meurisse, P. Mirchev, N. Nemer, M.-R. Paiva, J. Pino, A. Protasov, N. Rahim, J. Rousselet, H. Santos, D. Sauvard, A. Schopf, M. Simonato, A. Yart, M. Zamoum. 2015. Natural History of the Processionary Moths (Thaumetopoea spp.): New Insights in Relation to Climate Change. – In: Roques A. (Ed.) Processionary Moths and Climate Change: An Update. Springer, 15-79. EFA. 2010-2019. Executive Forest Agency. Report on the results of the Pathological surveys for pests, diseases and other damages in forests. http://www.iag.bg/docs/lang/1/cat/6/ index. Georgieva, M., G. Zaemdzhikova, P. Mirchev, G. Georgiev. 2019. Larval hatching dynamics of Thaumetopoea pityocampa summer phenological form in the Eastern Rhodopes, Bul- garia. – Forestry ideas, vol. 25, 2 (58), 451-458. Huchon, H., G. Démolin, 1970. La bioécologie de la processionnaire du pin. Dispersion po- tentielle – dispersion actuelle. – Revue Forestière Française, spécial ‘La lutte biologique en forêt’, 220–234. Masutti, L. 1964. Ricerche sui parassiti oofagi della Thaumetopoea pityocampa (Schiff.). – Annali del Centro di Economia Montana delle Venezie, 4, 205-271. Mirchev, P., G. Georgiev, A.Tashev. 2011a. Instar structure of caterpillars of pine proces- sionary moth in Bulgaria during the cold period of the year. – Forest science 1-2, 37-46. Mirchev, P., G. Georgiev, M. Matova. 2011b. Prerequisites for еxpansion of pine procession- ary moth Thaumetopoea pityocampa (Den. & Schiff.) in Bulgaria. – Journal of Balkan Ecology, 14, (2), 117-130. Mirchev, P., G. Georgiev, G. Geshev. 2013. Dispersion of Male Butterflies of Pine Proces- sionary Moth (Thaumetopoea pityocampa). – Silva Balcanica, 14 (1), 102-108 Mirchev, P., G. Georgiev, M. Georgieva, L. Bocheva. 2016. Impact of low temperatures on pine processionary moth (Thaumetopoea pityocampa) larval survival in Bulgaria. – Silva Balcanica, 17(1), 51-58.

108 Mirchev, P., G. Georgiev, M. Georgieva, M. Matova, G. Zaemdzhikova. 2017. Enlargement of the pine processionary moth (Thaumetopoea pityocampa) range in Bulgaria. – Forest review, 48 (1), 4-7. Mirchev, P., M. Georgieva, G. Zaemdzhikova, M. Matova, S. Hlebarska, E. Filipova, G. Georgiev. 2019. Phenological form diversity of Thaumetopoea pityocampa in Bulgaria. – Poplar, 203, 65-69. Santos, H., J. Rousselet, E. Magnoux, M.-R.Paiva, M. Branco, C. Kerdelhue. 2007. Genetic isolation through time: allochronic differentiation of a phenologically atypical population of the pine processionary moth. – Proceedings of the Royal Society, B 274, 935-941. Santos, H., C. Burban, J. Rousselet, J.-P. Rossi, M. Branco, C. Kerdelhue. 2011. Incipient allochronic speciation in the pine processionary moth (Thaumetopoea pityocampa, Lepi- doptera, Notodontidae). – Journal of Evolutionary Biology, 24 (1), 146-58. Santos, H., M.-R. Paiva, S. Rocha, C. Kerdelhu, M. Branco. 2013. Phenotypic divergence in reproductive traits of a moth population experiencing a phenological shift. – Ecology and Evolution, 3 (15), 5098-5108. Schmidt, G. H., J. Kitt 1994. Identification by meconia of two egg parasitoids Thaumetopoea wilkinsoni. – Phytoparasitica, 22 (1), 39-41. Schmidt, G. H., G. Tsankov, P. Mirchev. 1997. Notes on the egg parasitoids of Thaumeto- poea pityocampa (Den. & Schiff.)(Insecta Lepidoptera Thaumetopoeidae) collected on the Greek island Hydra. – Bollettino di Zoologia agraria e di Bachicoltura, Ser. II, 29 (1), 91-99. Skufin, K. 1949. Ecology of tabanids in the Voronezh Region. – Zoology Journal, 28, 145-156. (In Russian). Tanzen, E., G. H. Schmidt. 1995. Identification by meconia of four species egg parasitoids Thaumetopoea pityocampa (Den. & Schiff.) (Insecta Lepidoptera Thaumetopoeidae). – Bollettino di Zoologia agraria e di Bachicoltura, Ser. II, 27 (1), 61-70. Tsankov, G., G. H. Schmidt, P. Mirchev. 1996. Parasitism of egg-batches of the pine proces- sionary moth Thaumetopoea pityocampa (Den. & Schiff.) (Lep., Thaumetopoeidae) in various regions of Bulgaria. – Journal of Applied Entomology, 120, 93-105. Tsankov, G., G. H. Schmidt, P. Mirchev. 1998a. Distribution of egg parasitoids of the pine processionary moth Thaumetopoea pityocampa (Den. et Schiff.) (Lep., Thaumetopoeidae) in the southwestern region of Bulgaria. – Forest science, 3/4, 5-17. Zaemdzhikova, G., C. Robinet, M. Georgieva, G. Georgiev, L. Bocheva, G. Tsankov, A. Bernard, M. Buradino, J-P. Rossi, A. Roques, C. Kerdelhue, J. Rousselet, P. Mirchev, M. Laparie. 2018. Emergence of atypical PPM phenologies at both longitudinal ends of its northern expansion front: the importance of identifying local drivers. – Presentation to First Processionary Moth Meeting, Hammamet, Tunisia, 6-8 December 2018. Zankov, G. 1960. Untersuchungen über einige Merkmale aus der Biologie und Oekologie Kiefernprozessionsspinners (Thaumetopoea pityocampa Schiff.) bei uns in Verbindung mit den Methoden zu seiner Bekämpfung. – Ministerium für Land und Forstwirtschaft, Forschungsinstitut für Forst und Forstwirtschaft, Wissenschaftliche Arbeiten, Bd. VIII, 231-262. (In Bulgarian, German summary).

109 ВАРИАБИЛНОСТ ВЪВ ФЕНОЛОГИЯТА И ЕКОЛОГИЯТА НА БОРОВАТА ПРОЦЕСИОНКА (THAUMETOPOEA PITYOCAMPA) В ЮГОИЗТОЧНИТЕ РОДОПИ, БЪЛГАРИЯ

Г. Заемджикова, Г. Георгиев, М. Георгиева, М. Матова, П. Мирчев Институт за гората – София Българска академия на науките

(РЕЗЮМЕ)

Проведени са изследвания върху фенологията и екологията на боровата процесионка (Thaumetopoea pityocampa) в насаждения от черен бор (Pinus nigra) в четири землища в Югоизточните Родопи: Ивайловград, Кирково, Кандилка и Ауста. В находищата край с. Кирково и с. Кандилка е установена “лятната фенологична форма” на T. pityocampa. В находището край Ивайловград е установена “зимната фенологична форма” на вредителя, а в землището на с. Ауста се развиват заедно и двете форми, през зимните месеци е установено, че близо 20% от гнездата се напускат за хибернация в почвата, но в останалите – гъсениците остават и през зимата. В два обекта с различна фенологична форма – Ивайловград и Кирково, са изследвани яйчните паразитоиди на боровата процесионка, като не е установена разлика в паразитоидния комплекс при двете форми. Доминантен паразитоид и в двете местообитания е Baryscapus servadeii, следван от полифага Ooencyrtus pityocampae. Във всичките проби присъства хиперпаразитоидът Baryscapus transversalis, който опаразитява средно 5% от установените парзитоиди.

Key words: Thaumetopoea pityocampa, фенология, екология, България

Ел. поща: [email protected]

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