Silva Balcanica, 17(1)/2016

Impact of low temperatures on ( pityocampa) larval survival in

Plamen Mirchev1, Georgy Georgiev1, Margarita Georgieva1, Lilia Bocheva2 1 Forest Research Institute – , Bulgarian Academy of Sciences 2 National Institute of Meteorology and Hydrology – Sofia, Bulgarian Academy of Sciences

Abstract

Thaumetopoea pityocampa winter nests were collected in January 2011 and 2015 from six localities situated in different regions over pest’s range in Bulgaria. In each site, ten winter nests were randomly selected and the larval instar structure and their survival were determined. The ‘silk index’ of winter nests and the level of their building were assessed. Using data about average daily and minimal monthly temperatures from November (2010, 2014) to February (2011, 2015), the number of favourable days for T. pityocampa larval development was evaluated. A relationship between the climatic characteristics over the cold period of year and the larval instar structure, survival, adaptability expressed by the level of building winter nests and the time of nest leaving and moving of the larvae into the soil was found out. Key words: Thaumetopoea pityocampa, instar structure, larval survival, distribution, Bulgaria

INTRODUCTION

Pine processionary moth, Thaumetopoea pityocampa (Denis & Schiffermüller, 1775) (: , Thaumetopoeinae) is considered to be one of the most frequent and dangerous pests in the Mediterranean region. A part of the northern border of pest distribution passes through Central Bulgaria. In this area, the specific ecological form T. pityocampa bulgarica occurs as more adaptive to extreme climatic conditions than the typical Mediterranean form (Tsankov et al., 1996). The geographic range of T. pityocampa in Bulgaria covers pine forests in the Rhodopes Mt. and the mountainous areas of South Bulgaria, predominantly along Struma and Mesta river valleys. The border of its northern distribution reaches to Golo Bardo Mt., Sredna gora Mt. and to the south slopes of Central Balkan Range (Mirchev et al., 2011b). During the cold period, the survival of pine processionary moth caterpillars depends on the value of air temperature, which should not fall below -16ºС (Démolin, 1969b). In winter period, the most suitable for larval development are daily temperatures of over 9ºC followed by night temperatures above 0ºC allowing caterpillars to leave their nests and feed on the needles of the pine trees (Battisti et al., 2005). Milan (1990) reported that well-constructed nests protect the caterpillars against unfavourable climatic conditions.

51 The aim of this work was to investigate the main biological characteristics of T. pityocampa (larval instar structure, survival, adaptability expressed by the level of building winter nests, the time of nest leaving and moving of the larvae into the soil) in different regions of its range during the cold period of year (November-February) and relationship between these characteristics and temperature conditions.

MATERIALS AND METHODS

The biological material was collected from six localities over the pine processionary moth range in Bulgaria (Table 1). In one of them, Pesnopoy ( district), T. pityocampa bulgarica occurs and in the rest ones, the typical form is distributed. The locations Marikostinovo and Ivaylovgrad are situated in regions with Mediterranean climate influence where T. pityocampa occurs permanently. The climate of Pchelarovo is less influenced by Mediterranean climate than Ivaylovgrad in the Eastern Rhodopes. Staro selo is situated in a region with continental climate where T. pityocampa appears in some years. Muhovo occupies an intermediate position between Sofia and Plovdiv, with mild and warm microclimate determined by the influence of close Topolnitsa dam and south mountain slopes of Sredna gora. Climatic data (average daily and minimal monthly temperatures) for November and December (2010 and 2014), and January and February (2011 and 2015) for four of the investigated locations were obtained by the meteorological stations in , , Plovdiv and Sofia (Table 1). In 2011, T. pityocampa nests were collected from Austrian black pine (Pinus nigra Arn.) plantations in six sites to study the larval instar structure during the cold period (Mirchev et al., 2011a). In January 2015, the same places were visited again in order to collect biological material for new analyses (Table 1). In each site, ten winter nests were randomly selected and all available caterpillars in them were taken to determine the larval instar structure and their survival. The dead caterpillars were separated for a further microbiological analysis, and the living ones were placed in 75% ethanol. In the entomological laboratory of Forest Research Institute – Sofia, the larval developmental stage was determined by the size of head capsule (Zankov, 1960). The nests were assigned by nest silk index according to a three-stage scale (Battisti et al., 2005): 1 – loose nest wall, larvae visible through the wall; 2 – thick nest, larvae not visible, external silk layer sparse; 3 – very thick nest, larvae not visible, external silk layer dense. At recent study, the intermediate degrees of 1.5 and 2.5 were used. The data were analysed using descriptive statistic of MS Excel 2013. The relationship between the various variables was determined by the Pearson correlation coefficient (r).

RESULTS

T. pityocampa winter nests characteristics In 2015, with 60 analyzed nests, eight of them were empty because the caterpillars had already moved into the soil. Out of the rest 52 nests with caterpillars present, the

52 Table 1. Main characteristics of the studied areas and meteorological stations

Meteorological Date of collection station Geographical Altitude, Altitude, region Site Coordinates 2011 2015 Name m m - Marikosti- 41º26’24.5”N, 130 January 17 January 30 Sandanski 270 Sandanski novo 23º10’39.3”E kettle river Ivaylov- 41º31’40.0”N, valley, the 250 January 20 January 20 Kardzhali 290 26º07’07.3”E Eastern Rhodopes Central part 42º20’37.7”N, Pesnopoy 300 January 18 January 14 Plovdiv 130 of Thracian 24º40’15.1”E plain 42º28’49.4”N, Golo bardo Staro selo 830 January 21 January 30 Sofia 600 23º06’53.3”E Mt. Topolnitsa 42º25’10.0”N, river valley, Muhovo 400 January 18 January 14 - - 23º59’57.1”E Sredna gora Mt. Uplands of 41º46’47.9”N, Pchelarovo 480 January 20 January 20 - - the Eastern 25º21’27.1”E Rhodopes nest silk index was assessed at 2.0 or 3.0 degree in predominate part of them (28.9% and 26.9%, respectively), followed by the nest silk index degree of 2.5 (19.2%), 1.5 (15.4%) and 1.0 (9.6%). The number of caterpillars in different nests varied over a rather wide margin: from 8 in Ivaylovgrad to 568 in Pchelarovo. During both periods of study, the lowest average number of caterpillars in one nest was registered in the region of Pesnopoy where T. p. bulgarica occurs. The highest number was assessed in Pchelarovo and Staro selo (Table 2). No correlation (r = -0.05) was detected between the number of T. pityocampa caterpillars and the silk thickness of winter nests. Although in Ivaylovgrad and Staro selo it occupies habitats in two different climatic zones (Table 2), the nest silk index was similar in both studied periods. In contrast to them, the nest silk index was different in Ivaylovgrad (2.36-2.60) and Marikostinovo (1.85-2.15), situated in regions with expressed influence of Mediterranean climate and permanent presence of pine processionary moth in them. The data of study showed no correlation between the thickness of nests and caterpillars mortality (r = -0.12). Relationship between T. pityocampa caterpillars survival and climatic parameters over the winter period Feeding of T. pityocampa caterpillars mostly occurs at night. As was mentioned above, wintertime feeding is possible when night temperature is above 0ºC (potential

53 Table 2. Main characteristics of T. pityocampa studied nests

Insect Nest silk index Total number No. larvae/colony ± SD Locality generation ± SD of caterpillars (Range) 2010-2011 2.36±0.50 1115 111.50±54.91 (42-215) Marikostinovo 2014-2015 2.60±0.66 952 105.78±27.5 (17-450) 2010-2011 1.85±0.47 879 87.90±64.75 (8-229) Ivaylovgrad 2014-2015 2.15±0.53 1109 110.90±61.74 (19-238) 2010-2011 2.40±0.61 313 62.60±55.43 (10-135) Pesnopoy 2014-2015 2.0±0.58 355 71.00±34.50 (26-111) 2010-2011 1.68±0.26 927 115.87±46.87 (37-204) Staro selo 2014-2015 2.20±0.48 1288 128.80±57.28 (26-228) 2010-2011 2.40±0.57 908 90.80±76.27 (23-295) Muhovo 2014-2015 2.30±0.79 898 99.78±36.34 (50-147) 2010-2011 2.05±0.50 760 108.57±60.67 (20-188) Pchelarovo 2014-2015 2.00±0.85 1334 148.22±161.85 (29-568) feeding temperature) provided that the colony temperature during the day before has reached 9ºC (activation temperature). In respect to the number of favourable days for pine processionary moth caterpillars’ development, it was established that the most favourable conditions occurred during 2010-2011 in the region of all four meteorological stations ranging between 24 for Staro selo and 46 for Marikostinovo (Table 3). In 2014-2015, the number of favourable days for all stations was lower. In Sofia region it was less with 33.3% as compared to 2010-2011 and in for the rest stations the results were similar, respectively: Plovdiv – 61.1%, Kardzhali – 63.4% and Sandanski – 78.3%. As concerning to the average daily temperatures sum, these differences during the two studied periods were not noticed for all stations (Table 3). Critical for the caterpillars’ survival temperature values (below -16ºC) were not registered. Beside availability of pest host plant, the number of the favourable days in cold period is also a necessary condition one biotope to be appropriate for the pine processionary moth caterpillars survival and development. A relatively high negative correlation (r = -0.77) was established. It could be conditionally assumed that for 120- day period (from November to February) the rate of mortality increased significantly when the number of favourable days was below 15 (LD50= 14.39 days) (Fig. 1). It was also revealed that the survival of T. pityocampa caterpillars was less affected by the sum of average daily temperatures (r = -0.52) during the cold period (November to February). The relief characteristics and other environment components were significant for the specific microclimatic conditions. In Staro selo, on the slope with eastern exposition, it was detected that the highest caterpillars’ mortality occurred predominantly in the nests collected from the lower part of slope along a gully, most probably due to more severe wind currents and higher local humidity.

54 Table 3. Survival of T. pityocampa caterpillars and weather conditions during the winter period

Favourable days Σ of average daily Meteorological for winter feeding temperatures Mortality, Site Year station from November % n % to February 2010-2011 46 38.3 831.4 7.9 Marikostinovo Sandanski 2014-2015 38 31.6 768.0 17.9 2010-2011 41 34.0 688.7 0.8 Ivaylovgrad Kardzhali 2014-2015 26 21.7 643.7 28.7 2010-2011 36 30.0 535.1 5.8 Pesnopoy Plovdiv 2014-2015 22 18.3 593.8 11.8 2010-2011 24 20.0 311.2 12.1 Staro selo Sofia 2014-2015 8 6.7 332.3 85.7

In a half of studied nests in Pesnopoy (2011 and 2015), the caterpillars had already left their nest and moved into the soil for overwintering (Table 4). Lower values (10- 30%) of empty nests were registered in Muhovo and Pchelarovo locations, as well. In January, the caterpillars of T. pityocampa were at fourth-fifth instar in all studied localities. In 2010-2011 generation, the half part of nests in Pesnopoy was totally empty and in the rest all caterpillars completed their development, being at fifth instar (Table 4). In the same locality in 2014-2015 generation, 34.8% from detected caterpillars were at fourth instar because of less number of favourable days, as compared to ones during the previous generation. Similar situation was observed in Ivaylovgrad, where the caterpillars were at fourth instar and the period of favourable days in 2014-2015 was with 15 days shorter than in 2010-2011. The fluency of the number of favourable days on the instar structure cannot be absolutized because of the undoubtedly influences of other specific microclimate factors. Deviation of straight dependence between the number of favourable days and instar structure was determined at the material collected from Маrikostinovo and Staro selo. At the first location, despite of less number of favourable days in 2014-2015, the caterpillars’ development was faster and predominated ones at fifth instar as compared to the previous period. The small number of survived caterpillars in the region of Staro selo in 2014-2015 was with the same age structure, although the number of favourable days in 2010-2011was three times fewer.

DISCUSSION

In Bulgaria, the areas infested by pine processionary moth have expanded since 1975. In this period, two stages could be determined: i) the pest expansion was within the range and mainly due to the massive afforestation carried out and pine stands area has increased several times till the end of the twentieth century; ii) since 1999, the pest

55 Table 4. Larval instar structure and hibernation characteristics of T. pityocampa

Instar of caterpillars (%) Hibernation Average Locality Year colonies in the soil, instar 3rd 4th 5th (%) 2010-2011 0.1 72.9 27.0 4.27 0.0 Marikostinovo 2014-2015 0.1 54.3 45.6 4.46 0.0 2010-2011 0.0 0.2 99.8 5.00 0.0 Ivaylovgrad 2014-2015 0.0 82.8 17.2 4.17 0.0 2010-2011 0.0 0.0 100 5.00 50.0 Pesnopoy 2014-2015 0.0 34.8 65.2 4.65 50.0 2010-2011 7.6 91.9 0.5 3.93 0.0 Staro selo 2014-2015 0.0 98.9 1.1 4.01 0.0 2010-2011 0.0 52.9 47.1 4.01 0.0 Muhovo 2014-2015 0.0 48.7 51.26 4.47 10.0 2010-2011 0.0 1.1 98.9 4.99 30.0 Pchelarovo 2014-2015 0.0 59.2 40.8 4.41 10.0 has distributed to the east in new biotopes thoroughly in Sredna gora Mt. and on the southern slopes of Central Balkan Range (Mirchev et al., 2011b). The high number of T. pityocampa larvae established in some nests (up to 568) confirms the possibility of combining the caterpillars from several primary nests into one winter nest. In this aspect, it is not possible to talk about the generative potential, expressed by the female egg fecundity in the studied sites. The restricting effect of temperatures below -16◦С and the lack of favourable days or their limited number in the year, should be relatively taken into consideration. The ecological form T. p. bulgarica, unlike the Mediterranean one, completes its development till the commencement of the cold period and overwinters in the soil (Zankov, 1960; Tsankov еt al., 1996). In this study, T. pityocampa empty nests were established in three localities only: Pesnopoy (50%), Pchelarovo (10-30%) and Muhovo (10% in 2015) (Table 4). Muhovo and Pchelarovo localities are situated at distance about 40-60 and 80-100 km, respectively, from the region of Pesnopoy, where the ecological form of T. p. bulgarica occurs. This fact arises the question for a possible genetic exchange between both forms because of flying of male butterflies at a distance of 50 km (Mirchev et al., 2013). The analysis of long-term data of climatic parameters in Bulgaria that regulate the pine processionary moth survival and feeding during the winter, together with the similar conclusions proposed by Battisti et al. (2005), revealed that only the region of Central Northern Bulgaria is unsuitable for pest development due to pronounced continental climate and often critical low winter temperatures (Mirchev et al., 2011b). The favourable conditions for pine processionary moth development are limited by the high summer temperatures that could kill the significant number of eggs and young caterpillars (Robinet et al., 2007), whereas the caterpillars in fifth instar could survive at temperatures about 32◦ С without an increase of mortality (Breuer, Devkota, 1990).

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90

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40 Mortality,% 30

20

10

0 0 5 10 15 20 25 30 35 40 45 50 Favourable days

Fig.1. Relationship between survival of T. pityocampa caterpillars and number of favourable for feeding

days during the cold period (November-February)

Fig.1. RelationshipThe exponential between function survival ofobtained T. pityocampa in current caterpillars study and(Fig. number 1) is the of favourablemost suitable forfor feeding illustration days duringof pine the processionary cold period (November moth larval-February) survival during cold period of year. The low values of the independent variable (favourable days) are scarcely recorded but it is evident that with zero favourable days the mortality must be 100%. It is also evident that too big number of favourable days will no more have dramatic effect on the survival. Beside the average daily temperatures, the species survival is also defined by the influence of other factors in the different parts of one biotope. Breuer, Devkota (1990) revealed that there are different temperature parameters into the nest in depending on the lit by the sun. If the temperature in the nest increases up to 1.5◦ С above this in the environmental area, this rise in temperature is contributed by the biogenic heat production. The results of our study do not corroborate the theses of Milani (1990) and Breuer, Devkota (1990) for the protective function of well-built nests against unfavourable climatic conditions. The conclusions made by Demolin (1969a) and Geri (1983, 1984) that well-built nests could be made only by huge number of caterpillars were not confirmed by our results – no correlation between the number of caterpillars and the silk thickness (r = -0.05). In Pesnopoy, the half part of caterpillars had left the winter nests and moved in the soil in mid-January. It was established that a part of caterpillars (10-30%) in Pchelarovo and Muhovo, situated in regions with mountain influence, had left the winter nests. The presence of overwintering caterpillars in the soil during this period of year could be considered as extending of T. pityocampa bulgarica range because of proximity of these sites to its main distribution area. In Portugal, Santos et al. (2013) revealed that both summer and winter populations co-exist in one and the same pine stands. Staro selo is situated in the upper course of Struma river and is characterized with strong climatic

57 condition. In this location overwintering caterpillars were not established in the soil. In this region, during the years of favourable conditions, the Mediterranean ecological form of T. pytiocampa penetrated in pine plantations from South at middle course of Struma river when the species permanently occurs. The rate of pine processionary moth survival and overwintered caterpillars into the soil is a prerequisite to consider that since 1999 the detected pest’s expansion in Central Bulgaria has been continuing into new areas in the country. In 2015 it infested the green urban area ‘Tyulbeto’ in situated at a distance of 40 km from hills – the natural eastern border of its distribution till 1999, and therefore, T. pytiocampa expanded its range to the east by an average of 2.5 km per year.

REFERENSES

Battisti, A., M. Stastny, S. Netherer, C. Robinet, A. Schopf, A. Roques, S. Larsson. 2005. Expansion of geographic range in the pine processionary moth caused by increased winter temperatures. Ecological applications, 15 (6), 2084-2096. Breuer, M., B. Devkota. 1990. Studies on the importance of nest temperature of Thaumetopoea pityocampa (Den. & Schiff.) (Lep., Thaumetopoeidae). J. of applied entomology, 109, 331-335. Démolin, G. 1969a. Bioecología de la procesionaria del pino Thaumetopoea pityocampa Schiff. Incidencia de los factores climáticos. Boletín del Servicio de Plagas Forestales, 12, 9-24. Démolin, G. 1969b. Comportement des adultes de Thaumetopoea pityocampa Schiff. Dispersion spatiale, importance écologique. Annales Scientifiques Forestières, 1, 81-102. Geri, C. 1983. Distribution and evolution of populations of the pine processionary, Thaumetopoea pityocampa (Den. & Schiff.) (Lep., Thaumetopoeidae) in the Corsican mountains. I. Emergence rhythms of the insect and population dynamics. Acta Oecologica. Oecologia Applicata, 4, 247-268. Geri, C. 1984. Distribution and evolution of populations of the pine procrssionary, Thaumetopoea pityocampa Schiff. (Lep., Thaumetopoeidae) in the Corsican mountains. II. Relations between populations and the physical and forest environment. Acta Oecologica. Oecologia Applicata, 5 (1), 3-22. Milani, R. 1990. The temperature of the egg masses of Thaumetopoea pityocampa (Den. & Schiff.) (Lepidoptera, Thaumetopoeidae). Redia 73 (1), 149-161. Mirchev, P., G. Georgiev, A. Tashev. 2011a. Instar structure of caterpillars of pine processionary 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 Expansion of Pine Processionary Moth Thaumetopoea pityocampa (Den. & Schiff.) in Bulgaria. J. of Balkan Ecology, 14 (2), 117-130. Mirchev, P., G. Georgiev, G. Geshev. 2013. Dispersion of male butterflies of pine processionary moth (Thaumetopoea pityocampa). Silva Balcanica, 14 (1), 103-109. Robinet, C. P. Baier, J. Pennerstorfer, A. Schopf, A. Roques. 2007. Modeling the effects of climate change on the potential feeding activity of Thaumetopoea pityocampa (Den. & Schiff.) (Lep. Notontidae) in France. Global Ecology and Biogeography, 1-12. 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. Tsankov, G., G. H. Schmidt, P. Mirchev. 1996. Structure and parasitism of egg-batches of a processionary moth population different from Thaumetopoea pityocampa (Den. & Schiff.) (Lep. Thaumetopoeidae) found in Bulgaria. Bollettino di Zoologia agraria e di Bachicoltura, Ser. II, 28 (2), 195-207. 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).

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