ISSN 1302-0250

Journal of the Entomological Research Society ------Volume: 19 Part: 1 2017 JOURNAL OF THE ENTOMOLOGICAL RESEARCH SOCIETY Published by the Gazi Entomological Research Society

Editor (in Chief) Abdullah Hasbenli Managing Editor Associate Editor Zekiye Suludere Selami Candan Review Editors Damla Amutkan Doğan Erhan Ersoy Nurcan Özyurt Koçakoğlu

Subscription information Published by GERS in single volumes three times (March, July, November) per year. The Journal is distributed to members only. Non-members are able to obtain the journal upon giving a donation to GERS.

One issue price: Annual subscription price (3 issues,about 300 pp.) 5 (Turkey) 20 (Turkey) US$ 20.00 US$ 60.00

Papers in J. Entomol. Res. Soc. are indexed and abstracted in Biological Abstract, Zoological Record, Entomology Abstracts, CAB Abstracts, Field Crop Abstracts, Organic Research Database, Wheat, Barley and Triticale Abstracts, Review of Medical and Veterinary Entomology, Veterinary Bulletin, Review of Agricultural Entomology, Forestry Abstracts, Agroforestry Abstracts, EBSCO Databases, Scopus and in the Science Citation Index Expanded.

Publication date: August 05, 2017

© 2017 by Gazi Entomological Research Society

Printed by Hassoy Ofset Tel:+90 3123415994 www.hassoy.com.tr J. Entomol. Res. Soc., 19(2): 01-10, 2017 ISSN:1302-0250

Life Cycle and Host Preference of Xylotrechus rusticus (L., 1758) (Coleoptera: Cerambycidae) in the Heilongjiang Province, China

Tianzhong JING1 Xianhui HU2 Chunxiang HU3 Chengde LI4 Kuanyu LIU5 Zhiying WANG6*

1, 3, 4, 5, 6School of Forestry, Northeast Forestry University, Harbin 150040, CHINA 2Shunagcheng Forestry Bureau, Harbin 150100, CHINA e-mails: [email protected], [email protected], [email protected], [email protected], [email protected], *[email protected]

ABSTRACT The gray tiger longicorn beetle, Xylotrechus rusticus (L., 1758) (Coleoptera: Cerambycidae), is a wood-boring forest insect species. An outbreak of this insect in the 2000s caused a great deal of damage to the Three North Shelter Forests in the western part of Northeast China. To control this beetle and provide data for risk analysis, the development timing, life cycle and host preference were investigated. X. rusticus completed one generation per year and overwintered as larva in its last instar in Harbin, Heilongjiang Province, China. The sex ratio was biased 2:1 toward males. The natural mortality was only 4%. The diameter at breast height (DBH) of the infested poplar in this study ranged from 10 to 42 cm. The larvae crowded in the trunks at a height of 3-5 m above the ground. The percentages of attack among various poplar lines were significantly different. Key words: Gray tiger longicorn beetle, development timing, life history, host preference, sex ratio, poplar-boring longhorn beetle.

INTRODUCTION The gray tiger longicorn beetle, Xylotrechus rusticus (L., 1758) (synonym: Rusticoclytus rusticus (L., 1758)) (Coleoptera: Cerambycidae: Cerambycinae), was distributed in East Palaearctic, Near East (including Asian Turkey, Caucasian Russian republics, Georgia, Armenia, Azerbaidjan, Lebanon, Syria, Israel, Jordan, Sinai Peninsula (Egypt), Arabian peninsula, Iran, Iraq), North Africa (Not including Sinai Peninsula) and Oriental region (Sama, 2013). It attacks dead standings or fallen tree trunks (Brelih et al., 2006; Danilevskaya et al., 2009); the exception is in China, where it mainly attacks living poplar trees (Meng et al.,1983; Xu, 1979). The larvae dig tunnels in poplars, which can lead either to the break of the tree by wind or to its death (Fig. 1). The earliest documented outbreak of X. rusticus in Northeast China was reported in 1979 (Meng et al.,1983; Xu, 1979). We have recorded its recent outbreaks in the Heilongjiang Province. In 2003, Daqing Hongqi farm (Daqing City, Heilongjiang Province) performed clear cutting on 178 hm2 of forest that had been damaged by this beetle and additional selection cutting on another 186 hm2. From 2 JING, T., HU, X., HU, C. et al. 2000 to 2003, 129,798 trees (7,467 hm2) were cut in Shuangcheng (a new district of Harbin, Heilongjiang Province), and 200 hm2 of forest was cut in Zhaodong (Suihua City, Heilongjiang Province) due to the outbreak of this beetle. The outbreaks have also been reported in Changchun, the capital city of Jilin Province, in which the outbreak area spanned up to 1,300 hm2, 460 hm2 of which was seriously damaged (Meng, 2002).

Fig. 1. Damages of X. rusticus on poplar. (A) Trees broken by wind at the location of the infestation (white arrow head) and dead trees (black arrow head). (B) Debarking at the infested location on the trunk. (C) Emergence pores on the trunk. (D) Young larvae boring beneath the bark. (E) Old larvae and galleries burrowed in the xylem of poplars (black arrow head). (F) Galleries being filled with frass and dust (black arrow head). Though the serious damages caused by X. rusticus were only reported in China, this beetle is a potential threat to the rest of the world. Besides poplar, it also attacks species of Betula L., Salix L., Acer L., Castanea Mill., Quercus L., Sorbus L., Tilia L., and Ulmus L. (for review, see Han and Lyu, 2010) and even Abies alba L. (Szwalko and Krolik, 1990). Countries in which the tree taxa are distributed are potentially endangered areas, especially those in which X. rusticus is absent for now. For example, this beetle has been detected at the ports of USA (Berger, 2014), and is on the list of Indiana’s ‘most unwanted’ invasive plant pests (Indiana Cooperative Agricultural Pest Survey (CAPS) Program, 2015). The damaged sites in China are located in the central part of the Songnen Plain, and these forests are composed of single poplar lines. Poplar is distributed all over China (Chou et al., 1986). However, most poplar forests have been intentionally planted as wind breaking forest strips (shelterbelts). These strips of forests are known as the Three North Shelterbelt Program or the Green Great Wall. Poplar was selected to construct the Green Great Wall because it grows faster than other trees. Numerous hybrid poplar lines have been created in China to construct the Three North Shelter Forests. 3 Life Cycle and Host Preference of Xylotrechus rusticus An understanding of the biology and life cycle of this wood-boring beetle is important for determining appropriate control measures and their timing. However, the biology of this beetle is not very clear, even in China. In 2001-2002, we investigated the life history and other biological characters of this beetle in the Heilongjiang Province, China. These data will pave the way for the control of this beetle and provide information for risk analysis. In this study, we intended to investigate the preference of this beetle to poplar lines, providing references for stand regeneration. In addition, preferences for Diameter at Breast Height (DBH) and the height of the infestation on the trunk were also investigated, which is helpful to monitor and control this beetle.

MATERIALS AND METHODS

Life history survey The life history and habitat of the beetle were observed in the field in Shuangcheng (Fig. 2), now a district of Harbin, located in the central part of the Songnen Plain, China. The shelterbelts in Shuangcheng are composed of pure poplar stands. To determine the developmental timing of pupation and eclosion of X. rusticus, at least two infested trees (20-35 cm in DBH) (at least 60 insect specimens were collected) were cut into c.a. 100 cm long logs, and the logs were then chopped into pieces to record the numbers of larvae, pupae and adults in a survey. The insects from each log were also counted to plot the vertical distribution of X. rusticus on the trunk. The numbers of dead and live beetles were counted respectively to calculate the natural mortality which is equal to the number of total dead beetles divided by the sum of the numbers of total collected beetles. This survey started on May 23, 2001 and April 21, 2002 and was carried out every week until pupae were found. As soon as pupae were found, the investigation was conducted daily. All collected larvae and pupae were killed and buried after counting. To study the developmental timing of the beetle, trees must be felled and hewed. In China, there is a strict procedure to apply for permission to fell a tree. We did not apply for this permission. Alternatively, we conducted this investigation when the forest manager was doing a clear cut. All felled trees should be fumigated with chemicals before the beetles’ emergence in order to kill the beetles within the trunks. As a result, the dates we did the inspections were restricted to the timing of the clear cut. Since the dates for clear cut and fumigation varied from site to site, the complete investigations could not be carried out at all sites. With the permission and under the surveillance of the officials from the local forestry administrative organization, 30 logs were wrapped with wire cages (20 eye in mesh size) before emergence occurred, and the number and sex of the emerged beetle adults were recorded respectively every day to calculate the emergence rate and sex ratio. To avoid escaping, newly emerged beetles were killed by chemicals before they were collected out from the cage. After counting, the beetles were buried. This investigation lasted to another one week after no emergence occurred. Then all the logs were hewed and burned. 4 JING, T., HU, X., HU, C. et al.

Fig. 2. Study sites. Triangles represent the sites in Shuangcheng district. Generally speaking, the development of insects in any life stage can be sub-divided into starting period, peak period, and ending period when 16, 50, and 84% of the population reaches this stage, respectively (Xiao, 1992). There are two methods to determine these periods in the field. For emergence time, the chart of the cumulative emergence rate against date was plotted. However, this method is not suit for determination of the time of pupation and eclosion of this beetle since both the larvae and the pupae would die when they do not stay in the wood any longer. An alternative method was adopted to solve this problem. The daily percentages of pupation/ecolosion were plotted to make a normal-like curve. The date corresponding the vault of the bell curve is the peak period of pupation/eclosion.

The preference for poplar lines To verify whether X. rusticus has a preference for certain poplar lines, we investigated the attack percentages of six poplar lines at eight sites of the Shuangcheng District (Fig. 2). Poplar trees with similar DBH (ca. 20-35cm) were selected for investigation to avoid the influence of poplar age. If obvious characteristics of attack (e.g., emergence pore) were observed on a tree, it was assigned as an attacked one.

Statistics All statistical analyses and charts were designed using R (R Core Team, 2014). The study sites were mapped using the OpenStreetMap package (Fellows, 2013). Curve fit was carried out with nls function and proportion test with prop.test() function. A series of z tests were adopted to perform multiple comparisons between the natural attack percentages of poplar lines, and Bonferroni-Holm adjustments were used to adjust the significance values (Holm, 1979). Shapiro test was used to determine the normality, and Kruskal-Wallis test was used to test one-way ANOVA by ranks. A series of Wilcox tests were used to perform multiple comparisons if null hypothesis in Kruskal-Wallis test was rejected, and the significance values were also adjusted with Bonferroni-Holm adjustments. 5 Life Cycle and Host Preference of Xylotrechus rusticus Abbreviations PXH: Populus × xiaohei T.S. Hwang and Liang; PSI: Populus simonii Carrière; PBJ: Populus beijingensis W.Y. Hsu; SXZ: Populus × xiaozhuanica W.Y. Hsu and Liang; PSU: Populus suaveolens Fisch. ex Loudon; PPN: Populus pseudosimonii Kitag. × Populus nigra L.

RESULTS

Development timing The pupation of X. rusticus in 2002 began between May 5 and May 10. The exact date was not determined for the data that were missing (Fig. 3A). May 12 was observed as the ending point of larva and the starting point of pupa, at which the larval population was equal to that of pupa (Fig. 3A). Afterwards, the larval population being less abundant than that of pupa and ended at May 21, while the pupal population went to its summit around at May 19, then went down. The peak period of pupa of X. rusticus fell in May 24 in 2001 (Fig. 3B), five days later than in 2002. May 31 was observed as the point when the larval population was equal to that of the adult (Fig. 3B). The eclosion began on May 26 in 2001, six days later than in 2002 (May 20) (Fig. 3A). The first appearance of emergence was observed on May 30 in 2001. It took four, six and seven days to reach its starting period (June 3 2001), peak period (June 5 2001) and ending period (June 6 2001), respectively (Fig. 5B). The cumulative emergence rate by date could be described as a Weibull curve: y = 1-exp(-0.0000452x5.31), where y is the cumulative emergence rate, and x is the number of days after May 29, with a range of (1, 8).

Fig. 3. Development time of X. rusticus in Shuangcheng in 2001 and 2002. (A) Pupation time in 2002. (B) Eclosion time and emergence time in 2001. The digits above bars show the numbers of insect specimens collected from wood per day. The data of emergence in (B) are cumulative emergence rates based on 676 insect specimens. Data from May 5-10, 15, 16, 18-20, 22, 24 in (A) and data from May 26 and June 3 in (B) are missing.

Life cycle Based on the data from field survey, we summarized the life cycle of X. rusticus as follows. In the Shuangcheng District, X. rusticus completed one generation per 6 JING, T., HU, X., HU, C. et al. year. The larvae in their final instar overwintered in the tunnels they had burrowed in the xylem of poplars. The overwintered larvae resumed boring in April of the following year. The tunnels were irregular, with many twists and turns. The larvae then entered the pupation stage in the middle of May. Before pupating, the larvae turned and made their way toward the exterior bark of the infested tree, and then made a pupal chamber in the outer sapwood, so that the adults could easily chew a round hole to emerge after the eclosion. Emergence occurred in late May. Adults mated as soon as they emerged from the trees. The eggs were laid into the bark crevices in clusters, which were comprised of ten to dozens of eggs. Larvae appeared in early June, initially burrowing into the bark, and entering the xylem in mid-August. The galleries were filled with a mixture of frass and shredded wood. In late October, larvae entered into the heartwood to overwinter. The life cycle is shown in Fig. 4.

Sex ratio Of the 676 beetle specimens collected from 30 logs, 234 of them were females and 442 were males, fitting a ratio of 1:2 (female: male) (Chi-squared = 0.444, df = 1, p-value = 0.5052).

Natural mortality Totally 1,393 beetle specimens were collected from hewed wood in the survey of 2001(May 23 to June 2), and 54 (40 larvae and 14 pupae) of them were dead and the rest were live (92 larvae, 863 pupae and 384 adults). Thus, the rate of mortality was 3.88%; proportion testing showed that the mortality equals 4% (Chi-squared = 0.0278, df = 1, p-value = 0.43), with a 95% confidence interval of (0.00, 4.86). The mortalities of the larva and pupa were 43.48% and 1.62%, respectively. Proportion testing showed that the mortalities of the larva and pupa were equal to 50% and 2%, with 95% confidence intervals of [0.00, 52.58] and (0.00, 2.57), respectively. Thus, the mortality of the larva was 25 times higher than that of pupa. The mortality of the adults was not observed. The death of insects caused by diseases and parasitoids was not observed.

Fig. 4. Life history of X. rusticus in the Shuangcheng District. 7 Life Cycle and Host Preference of Xylotrechus rusticus Host preference The DBH of the damaged trees in this study ranged from 10-42 cm, with a mean of 27.51±6.51 cm (mean±SD) (Fig. 5A). The median numbers of beetles per m on trunk from 1-9 m above the ground were 0, 0, 16, 15, 24, 0, 0, 0 and 0, respectively, indicating that the majority of the larvae were burrowed into the trunks at a height of 3-5 m above the ground (Fig. 5B). The numbers at 3 m, 4 m and 5 m were used to perform statistical analysis. Shapiro test showed that the numbers do not have a normal distribution (W = 0.8649, p-value = 9.057e-05). Kruskal-Wallis test showed that the numbers of beetles per m on trunk from 3-5 m above the ground were not significantly different (Kruskal-Wallis Chi-squared = 0.1651, df = 2, p-value = 0.9208). The attack percentages of six poplar lines (Populus × xiaohei T.S. Hwang and Liang; Populus simonii Carrière; Populus beijingensis W.Y. Hsu; Populus × xiaozhuanica W.Y. Hsu and Liang; Populus suaveolens Fisch. ex Loudon; Populus pseudosimonii Kitag. × Populus nigra L.) ranged from 36-60% (Fig. 5C). P. × xiaohei had the lowest attack percentage, followed by P. simonii, P. beijingensis, P. × xiaozhuanica , P. suaveolens and P. pseudosimonii × nigra. A proportion test (z test) showed that there were significant differences in the percentages of the attack among these lines (Chi-squared = 33.8152, df = 5, p-value = 2.591e-06). The results of a series of multiple comparisons with Bonferroni-Holm adjustments in significant levels showed that the attack percentage of P. × xiaohei is statistically different from P. × xiaozhuanica , P. suaveolens and P. pseudosimonii × nigra, P. simonii from P. suaveolens and P. pseudosimonii × nigra, and P. beijingensis from P. suaveolens and P. pseudosimonii × nigra (Fig. 5C).

DISCUSSION AND CONCLUSIONS In this paper, we presented the life cycle (Fig. 4) and the timing of development of X. rusticus (Fig. 3), which are helpful to develop a control calendar. We started the survey on May 23 in 2001 since we did not have any information about the development time of this beetle. The results showed that the beetle was already in the peak period of pupa at that time (Fig. 3B). Therefore next year we started survey earlier (on April 21). As multiple sizes of the larvae were not observed during the investigation, it is reasonable to conclude that this beetle completes a generation in one year combined with the development time, as presented in Fig. 3. The peak period of pupa in 2002 was five days earlier than in 2001, indicating that the development time could be affected by the climate. The mean annual temperature (from previous July to current June) in 2001 was 0.527°C, while was 2.969°C in 2002. The mean annual temperature of 2001 was also far lower than that of 2000 (1.305°C). These data indicate that the extreme cold weather led to the development of X. rusticus postponed by five days. The peaks of the eclosion and emergence were five days apart (May 31 and June 5), suggesting that the adults do not emerge as soon as they eclose. According to the life cycle (Figs. 3 and 4), the best time to treat the adult beetles for the control should be in early June, and the best time to fumigate the larvae should be before May in the Shuangcheng District. 8 JING, T., HU, X., HU, C. et al.

Fig. 5. Host preference of X. rusticus. (A) Box-plot of preference for diameter on breast height ( DBH). (B) Box-plot of preference for height on the trunk. (C) Bar-plot of preference for poplar lines. “n” represents the total number of trees surveyed from each poplar line. The lowercase letters above bars in (C) show the significant differences. If two bars have a same letter, there are no significant differences in attack percentages between them and vice versa. The abbrevations of poplar line names see the Materials section in the text. Sex ratio is a basic characteristic of an insect population. Manipulation of sex ratio can be a method to control pests. We collected all the specimens of X. rusticus from 30 logs to calculate the sex ratio, equivalent to collecting beetles from the whole flight season. Our results showed that the sex ratio of the population in Shuangcheng was biased 2:1 toward males. The natural mortality of this beetle (excluding the stages of egg and adult after emergence) was low (4%). Lower mortality is a primary characteristic of an insect with K-selected history strategy (Pianka, 1970). The natural mortality of X. rusticus is even lower than that of another poplar-boring longhorn beetle, Anoplophora glabripennis (Motschulsky, 1853) (28%) (Zhao et al., 1993). The reason for this difference may be due to the galleries ofX. rusticus being filled with frass and dust, while the frass is extruded from the galleries of A. glabripennis. Blocked galleries prevent the entrance of natural enemies (especially parasitoids) and lead to difficulty in controlling insects. This reason also accounted for that none of parasitoids were observed in the field survey. Although the ground mortality was low forX. rusticus, the mortality of the larvae was as high as 50%. These results suggested that parasitoids are probably not an effective factor to control larvae or pupae of X. rusticus, while pathogens may be effective, especially for the larvae control. Unfortunately, the pathogens were not determined further. Our investigation showed that the DBH of attacked poplar trees ranged from 10-42 cm, suggesting that the beetle does not attack young trees. In this study, the larvae crowded in tree trunks at the height of 3-5 m (Fig. 5B). We also cut 100 poplar trees 9 Life Cycle and Host Preference of Xylotrechus rusticus into 50 cm-long logs, labeled the height on the trunk of each log, and then investigated the frequency of the attacked logs (Hu et al., 2004). The results showed that the height of 1-4 m above the ground on the trunk was attacked the most. These results showed that the larvae do not disperse to distribute uniformly on the trunk, even in high densities (the maximum was over 100 insect specimens per m; Fig. 5B). This habit led to the trees being broken by wind at the locations where the larvae were assembled. Meng et al. (1983) investigated the deposition heights on 62 trunks. In total, 186 egg clusters were found, 32% of which were on the lower parts of the trunks, 43% on the middle parts and 25% on the upper parts. These results suggest that the height of the infestation on the trunk is determined by the adults and the larvae disperse in quite a limited range. Silviculture is an important part of an integrated pest management program. Both stand structures and tree species composition are two primary factors determining the forest susceptibility to a forest insect pest (Loreau et al., 2001). The majority of poplar stands in China are single-species stands. It is well known that single-species stands are more susceptible to insects than mixed-species forests of more than 2-3 species (Loreau et al., 2001). It seems a good idea to afforest single-species stands with a less susceptible poplar species to insects. It is readily apparent from Fig. 5C that the natural attack percentages among the poplar lines were significantly different. The percentages of attack on P. × xiaohei and P. simonii lines were below 40%, suggesting that they are less susceptible to the beetle than the other lines and could be candidate species to afforest or reforest cutting blank. However, the preference of the insect for the host is relative. Planting a species susceptible to the insect (P. suaveolens or P. pseudosimonii × nigra), as a bait, together with less susceptible species (P. × xiaohei or P. simonii) will also be reasonable afforesting strategy. To sum up, X. rusticus has a potential expanding trend on the context of economic globalization, although it was not worldwide distributed for now. In this paper, we have presented data on the life history and host preference of this beetle, which are helpful to monitor and control this beetle globally.

ACKNOWLEDGEMENTS This study was financially supported by the National Natural Science Foundation of China (Grant No. 31370591), the Fundamental Research Funds for the Central Universities (Grant No. DL13CA07) and the Programs for Science and Technology Development of the Heilongjiang Province (GB01B203-02). We also acknowledge the assistance of the undergraduate students Minhui Xia, Renhu Yang, Jinde Ji and Liming Zhang in the field survey.

REFERENCES

Berger, P., 2014, APHIS Programs in Tree and Forest Health. http://www.continentalforestdialogue.org/ events/dialogue/2014-11-03/presentations/BERGER--Emerging-Regional-Issues.pdf. (11.03.2014) 10 JING, T., HU, X., HU, C. et al.

Brelih, S., Drovenik, B., Pirnat, A., 2006, Material for the beetle fauna (Coleoptera) of Slovenia. 2nd contribution: Polyphaga: Chrysomeloidea (= Phytophaga): Cerambycidae. Scopolia, 58: 1-442. Chou Y.-L., Tung S.-L., Nie S.-Q., 1986, Ligneous Flora of Heilongjinag (in Chinese). Heilongjiang Science Press, Harbin, China, 85. Danilevskaya, G. B., Danilevsky, M. L., Hadulla, K., Shapovalov, A. M., Yokoi, Y., 2009, Cerambycidae collected in North-East Kazakhstan by an international collecting trip 2005 (Coleoptera). Entomologische Zeitschrift, Stuttgart, 119: 171-178. Fellows, I., 2013, OpenStreetMap: Access to Open Street Map Raster Images. R Package Version 0.3.1. Using the JMapViewer Library by Jan Peter Stotz. http://CRAN.R-project.org/ package=OpenStreetMap. (01.31.2015) Han, Y., Lyu, D., 2010, Taxonomic review of the genus Xylotrechus (Coleoptera: Cerambycidae: Cerambycinae) in Korea with a newly recorded species. Korean Journal of Applied Entomolology, 49: 69-82. Holm, S., 1979, A simple sequentially rejective multiple test procedure. Scandinavian Journal of Statistics, 6: 65-70. Hu, C. X., Huang, Y. H., Li, C. J., Sun, J. H., 2004, Larval spatial distribution pattern of Xylotechus rusticus (in Chinese). Entomological Knowledge, 41: 241-244. Indiana Cooperative Agricultural Pest Survey (CAPS) Program, 2015, Indiana’s ‘Most Unwanted’ Invasive Plant Pests. http://extension.entm.purdue.edu/CAPS/insects.html. (06.26.2015) Loreau M., Naeem S., Inchausti P., Bengtsson J., Grime J. P., Hector A., Hooper D. U., Husto, M. A., Raffaelli D., Schmid B., Tilman D., Wardle D. A., 2001, Biodiversity and ecosystem functioning: Current knowledge and future challenges. Science, 294: 804-808. Meng, X. Z., 2002, Technological research of chemical prevention of Xylotrechus rusticus (in Chinese). Journal of Changchun University, 12: 12-14. Meng, Y. X., Zhang, M. J., Zheng, B. L., Wang, J., 1983, Biological characteristics and control of Xylotrechus rusticus (in Chinese). Jilin Forestry Science and Technology, 2: 28-33. Pianka, E. R. 1970, On r- and K-selection. American Naturalist, 104: 592-597. R Core Team, 2014, R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/. 01.31.2015 Sama, G., 2013, Fauna Europaea: Cerambycidae. In: Audisio, P. (Ed), Fauna Europaea: Coleoptera 2. Fauna Europaea version 2.6.2. http://www.faunaeur.org/. (01.31.2015) Szwalko, P., Krolik, R., 1990, The common fir (Abies alba Mill.), a new host plant for Xylotrechus rusticus (L.) (Col., Cerambycidae). Przeglad Zoologiczny, 34: 161-166. Xiao, G., 1992, Forest Insects of China (2nd Edition) (in Chinese). Forestry Publishing House of China, Beijing, 70. Xu, G., 1979, Preliminary study of Xylotrechus rusticus (in Chinese). Scientia Silvae Sinicae, 2: 159. Zhao, R., Lu, Z., Lu, X., Wu, X., 1993, Life table study of Anoplophora glabripennis (Coleoptera: Cerambycidae) natural populations (in Chinese). Journal of Beijing Forestry University, 15: 125-129.

Received: September 09, 2015 Accepted: October 21, 2016 J. Entomol. Res. Soc., 19(2): 11-22, 2017 ISSN:1302-0250

Review of the Phaonia consobrina Group (Diptera: Muscidae) from China, with Descriptions of Three New Species

Wanqi XUE1* Teng YU2

1,2Institute of Entomology, Shenyang Normal University, Shenyang 110034, CHINA e-mails: 1*[email protected], [email protected]

ABSTRACT The P. consobrina group from China is studied, and three new species are described: P. curvisetata, sp. n., P. niximountaina, sp. n. and P. subpilipes, sp. n.. A key to the identification of males of the 19 species of Palaearctic and Oriental zoogeographic regions and distributions are also given. Key words: China, Diptera, Muscidae, P. consobrina group, new species, key.

INTRODUCTION Robineau-Desvoidy (1830) established Phaonia, with P. viarum as the type species. The Phaonia species from the Palaearctic Region have been divided into 21 groups (Ringdahl, 1949; Hennig, 1963; Zinovjev, 1981). Ma et al. (2002) divided Chinese species of the genus Phaonia into 38 groups. P. angelicae group, firstly presented by Ringdahl (1949), was divided into three subgroups by Zinovjev (1981) and Xue (2006) revised P. consobrina subgroup as P. consobrina group. P. consobrina group is similar to P. angelicae group, but species of P. consobrina group are different from the latter that cerci are deeply concave in the middle. So P. consobrina group is distinguished by its basisternum of prosternum being bare; majority of male frons is narrower than twice of postpedicel width, without ors; epistoma protruded from frontal angle in profile; scutellum is black, without yellow area; notopleuron with hairs; mid tibia with 2 rows of p, hind tibia without apical pv; abdomen without yellow area; the male cerci are concave deeply in posterior view (Fig. 1), specialization distinct. Genus Phaonia has more than 820 species that are known all over the world. The habitat of most species in Phaonia consobrina group are found at alpine and cold areas, also a small group, consisting of only 19 species are found in Palaearctic and Oriental zoogeographic regions, including 17 species in China. In this paper, three new species are descriped, as well ass the key for these species and the distribution of known species are put forward.

MATERIALS AND METHODS The specimens for this paper by were examined sweeping brushwood in mountainous regions of northeast, southeast and central China. Genital structures were detached 12 XUE, W., YU, T. from the body, cleared by warming in a 10% NaOH solution (approximately 120°C) for several minutes, placed in a droplet of glycerol and observed under a compound light microscope. The type specimens of three new species are all deposited in the Institute of Entomology, Shenyang Normal University, Shenyang, China.

Fig. 1. The characteristic of male cerci of Phaonia consobrina group in posterior view. The morphological terminology follows that of McAlpine (1981). Absolute measurements are used for the body length in millimeters (mm). The following abbreviations are used for characters: ors (orbital setae), fr (frontal setae), prst acr (presutural acrostichal setae), acr (acrostichal setae), dc (dorsocentral setae), post dc (postsutural dorsocentral setae), ial (intra-alar setae), pra (prealar setae), av (anteroventral setae), ad (anterodorsal setae), pd (posterodorsal setae), p (posterior setae), pv (posteroventral setae), v (ventral setae), r-m (radio-medial cross-vein), dm-cu

(medio-cubital cross-vein), R4+5 (branch of radius), M (medial vein), Mt. (Mountain). Key to species of P. consobrina group from Palaearctic and Oriental (♂♂) 1. Hind tibia without additional pd subbasally…………….……….....…………2 -Hind tibia with additional pd subbasally………………….…….…...... ….…14 2. Post dc 3……………………………………………………………………3 -Post dc 4…………...…………………………………………………………8 3. Katepimeron bare……….…………….…Phaonia subnudiseta Xue, 1996 -Katepimeron with hairs……………………………………………………4 4. Frons slightly wider than anterior ocellus………………………………… ….……………………………………Phaonia angustinudiseta Xue, 1996 -Frons at least as wide as postpedicel……………………………………5 5. Eyes sparsely covered with ciliae, fronto-orbital plate upper with 2 ors… ………………………………………….…Phaonia grunnicornis Xue, 1996 -Eyes covered with thick ciliae, fronto-orbital plates without ors…………6 6. Frontal vitta twice as wide as fronto-orbital plates; hind tibia with 3 av … ……………………………………………Phaonia nudiseta (Stein, 1907) -Frontal vitta disappeared in the middle, with a sew only; hind tibia with 4 av...... 7 13 Review of the Phaonia consobrina Group from China 7. Calypters white and slightly yellow, halter brown yellow; fore tibia with 1-2 p, mid tibia with 2 ad………………Phaonia hirtirostris (Stein, 1907) -Calypters blackish, margin of calypter and halter black; fore tibia with 6-7 p, mid tibia with 4 ad…Phaonia nigrinudiseta Xue and Zhang, 1996 8. Fore tibia without medial p, hind tibia red-brownish, half of distal hind tibia with 2 pd, which are equal in length…………………………………… ……………………………………Phaonia dupliciseta Ma and Cui, 1992 -fore tibia with medial p, hind tibia black, with 1 pd or absent……………9 9. the longest arista hair at most half of postpedicel width………………10 -the longest hair length at least two thirds of postpedicel width………12 10. Mid tibia without ad, frons subequal in width to anterior ocellus………… ...... Phaonia niximountaina Xue and Yu, sp. n. -Mid tibia with ad 1, frons at least twice as wide as anterior ocellus.…11 11. The longest arista hair length one fifth of postpedicel width, frons twice as wide as anterior ocellus, calypters white to light yellow, halter brown, hind tibia with 3 av………….……Phaonia curvisetata Xue and Yu, sp. n. -The longest arista hair length half of postpedicel width, frons subequal in width to outer margin of posterior ocellus, calypters yellow, halter orange, hind tibia with 2 av……Phaonia paucispina Feng and Cui, 1988 12. parafacial slightly wider than postpedicel; calypters brown……………… ………………………………Phaonia cercoechinata Fang and Fan, 1986 -parafacial 1.5 times as wide as postpedicel, calypters dirt white……… ……………………………Phaonia cercoechinatoida Feng and Ma, 2002 13. Halter yellow, fronto-orbital plate adjoined in medial…...... …...... Phaonia consobrina Zetterstedt, 1838 -Halter brownish black, fronto-orbital plate not adjoined in medial…...... ……….Phaonia cercoechinatoida Feng and Ma, 2002 14. Post dc 3………………...... ………………Phaonia subpilipes Xue and Yu, sp. n. -Post dc 4………….…………...... ………………….……………………15 15. Fore tibia with 1 medial p…..………Phaonia pilipes Ma and Feng, 1986 -Fore tibia with 2-3 medial p……………...... …………………………16 16. Scutum only with one inconspicuous medial vitta...... ……...... ………..Phaonia spuripilipes Fang and Fan, 1992 -Scutum covered with 4 distinct black vittae…………...... …………17 17. Frons about equal to postpedicel in width...... Phaonia gergetica Zinovjev, 1994 -Frons about equal to half of postpedicel in width…...... 18 14 XUE, W., YU, T. 18. Frons equal in width to anterior ocellus, prementum 4 times as long as height, abdomen sparsely covered with pruinosity…...... ………...... Phaonia holcocerca Feng and Ma, 2000 -Frons equal in width to or slightly narrower than twice of anterior ocellus, prementum 3 times as long as height, abdomen covered with dense pruinosity……………………..……..Phaonia hesperia feng, 2004

Descriptions of new species

Phaonia curvisetata Xue and Yu, sp. n. (Figs. 2-5)

2 3

4 5

Figs. 2-5. Phaonia curvisetata Xue and Yu, sp. n. (holotype). 2. Male abdomen (dorsal view). 3. Male sternite 5 (ventral view). 4. Male cerci (posterior view). 5. Male terminalia (profile view). Scales: 2, 1.0 mm; 3, 0.5 mm; 4, 0. 5 mm; 5, 0. 5 mm. Male: Body length 7.1-8.1 mm. Head: Eyes covered with thick medium long brownish-yellow ciliae; frons about twice as wide as anterior ocellus, fronto-orbital plates touching, frontal vitta just situated one third of prementum; fr 10-11 pairs, expending to both sides of anterior ocellus, the upper 3-4 pairs short and small, without ors; fronto-orbital plate and gena covered with light grey pruinosity, parafacial wide, covered with copper-grey pruinosity, about 1.5 times as wide as postpedicel; antenna black, postpedicel 1.8-2.0 times as long as width, arista short haired, the longest hairs shorter than basal diameter of arista; facial carina low, vibrissal angle situated in front of frontal angle in profile; anterior margin of gena with 1-2 rows of subvibrissal setae, gena as high as about two fifths of eye, genal and postgenal hairs entirely black; proboscis long, prementum sparsely covered with pruinosity, 5.0 times as long as wide, palpus thin and long, slighty shorter than 15 Review of the Phaonia consobrina Group from China the length of prementum, labellum long, about twice as long as prementum, without distinct prestomal teech. Thorax: Black in background color, covered with light-grey pruinosity; scutum with 4 black vittae, the inner vittae not reaching scutoscutellar suture; scutellum black; acr 0+1, dc 2+4, ial 0+2, pra long and strong, about twice as long as posterior notopleural seta; notopleuron and katepimeron with hairs, the lower and flank of scutellum, basisternum of prosternum and meron all bare; katepisternal setae 1+2, spiracle big, anterior spiracle brown, posterior spiracle dark brown. Wings: Brownish, with basal of wings brown, wing veins fuscous, basicosta black, costal spine slightly shorter than length of r-m, dorsal and ventral of radial node bare,

R4+5 and M straight in terminal, the surroudings of r-m and dm-cu not clouded; calypters white to light-yellow and haltere brown. Legs: Entirely black; fore tibia with 2 medium p and 1-2 pv, mid femur without distinct av, pv row complete, 5-6 setae strong on basal half, mid tibia with 1 ad and 3-5 pd, sub-medium pv 1; hind femur with complete av row, becoming long and strong apically, 5-6 long and strong pv on basal half; hind tibia with 3 av, 5-6 ad in basal two thirds, 2 setae strong in distal, 1 pd in sub-basal, 5-7 setae behind the medium of hind tibia, without apical pv; claws and pulvillus long and strong, subequal in length to fifth tarsomere, middle and hind legs slightly short, subequal in length to fourth tarsomere. Abdomen: Black, sparsely covered with blue-grey pruinosity, tergite 3 with wide laddered patches, tergite 4 with black regular triangled patches, tergite 5 with black medial vitta, both sides without shining patch, vitta gloss black, sternite 1 bare. Female: Unknown. Etymology: The specific name refers to the male cerci with most setae curve. Diagnosis: This new species is similar to P. paucispina Feng and Cui, 1988, but differs from it in male characters: frons about twice as wide as anterior ocellus (frons as wide as outer margin of posterior ocellus in P. paucispina); wing brown on base (wing yellow on base in P. paucispina); halter brown (halter red yellow in P. paucispina); hind femur with 5-6 long and strong pv on basal half (hind femur with a row of pv completely in P. paucispina), cerci setae fringe-shaped curve (cerci setae stright in P. paucispina), anterior margin of cersus being curved backward in profile (anterior margin of cersus slightly stright in profile in P. paucispina), distal of free part wide (distal of free part thin and long in P. paucispina), inner margin without hair (inner margin with hairs in P. paucispina), posterior margin of surstylus curve in profile (posterior margin of surstylus stright in profile in P. paucispina). This new species is also similar to P. niximountaina, sp. n., but it differs from the latter in male characteristics: frons about twice as wide as anterior ocellus (frons subequal in width to anterior ocellus in P. niximountaina), gena about two fifths of eyes in height (gena about one fourth of eyes in height in P. niximountaina), prementum 5.0 times as long as width (prementum 4 times as long as width in P. niximountaina), calypters white to light yellow (calypters yellow in P. niximountaina), mid femur without distinct av (mid femur with av row in P. niximountaina), mid tibia ad 1 (mid tibia without ad in P. niximountaina), hind tibia 16 XUE, W., YU, T. with 5 to 6 ad in basal two thirds (hind tibia with 2 ad in P. niximountaina), distal of cersus wide (distal of cersus narrow in P. niximountaina), anterior margin of cersus being curved backward in profile (anterior margin of cersus slightly stright in profile in P. niximountaina), posterior of surstylus without hairs (posterior of surstylus with hairs in P. niximountaina).

Material examined: Holotype. 1♂ (IESNU), China, Mount Moirigkawagarbo (27.0237º N, 99.2607º E; 2500 m), Yunnan Province, 2500 m, 30.05.2013, Yu Teng. Paratypes. 20♂♂ (IESNU), same data as holotype; 9♂♂ (IESNU), China, Mount Baimang, Yunnan Province, 4400 m, 30.05.2013, Sun Hongkui; 8 ♂♂ (IESNU), China, Mount Baimang, Yunnan Province, 4400 m, 30.05.2013, Zhang Xiang.

Phaonia niximountaina Xue and Yu, sp. n. (Figs. 6-13)

6 7 8

9 10 11 12 13

Figs. 6-13. Phaonia niximountaina Xue and Yu, sp. n. (holotype). 6. Male abdomen (dorsal view). 7. Male sternite 5 (ventral view). 8. Male cerci (posterior view). 9, Male terminalia (profile view). 10. Female, spermatheca. 11. Female, sternites 1-5. 12. Female, ovipositor (dorsal view). 13. Female, ovipositor (ventral view). Scales: 6, 1.0 mm; 7, 0.5 mm; 8, 0.5 mm; 9, 0.5 mm; 10, 0.2 mm; 11, 1.0 mm; 12, 1.0 mm; 13, 1.0 mm. Male: Body length: 7.8-8.2 mm. Head: Eyes covered with thick long brown ciliae, frons subequal in width to anterior ocellus, fronto-orbital plate touching in middle, fr 11-12 pairs, reaching level of anterior ocellus, 5 short pairs of fr on upper half, subequal in length to ciliae of eyes, without ors; fronto-orbital plate, parafacial and gena covered with brown pruinosity, parafacial with a big golden-yellow patch at base of antenna, parafacial about 1.5 times as wide as flagellomere 1; antenna black entirely, flagellomere 1 about 1.8-2.0 times as long 17 Review of the Phaonia consobrina Group from China as wide, arista with short ciliae, longest hairs about 1.5 times as long as width of basal diameter of arista; epistoma situated in front of frontal angle, anterior margin of gena with 2-3 rows of subvibrissal setae; gena about one fourth of eye height, hairs on dorsal occiput and postgena black; prementum pruinose, 4.0 times as long as wide; palpus black, longer than prementum. Thorax: Black in background, sparsely covered with grey pruinosity, scutum with 4 wide black vittae, 0+1 acr, 2+4 dc, 0+2 ial, pra subequal to posterior notopleural seta, scutellum black, lateral and lower scutellum bare, notopleuron and katepimeron with hairs, basisternum of prosternum, anepimeron and meron all bare, spiracles dark brown, 1+2 katepisternal. Wings: Veins brown, basicosta black; costal spine short and small, radial node bare, surrouding of r-m and m-m distinctly clouded; r4+5 and m1+2 straight,­­ calypter yellow and halter dark brown. Legs: Entirely black, fore tibia without long ventral hair, 2 p, 2 pv, apical pv strong, as long as two thirds of tibia length, mid femur with haired av row, pv row complete and long, mid tibia without ad, but with 4 p, 1-3 pv; hind femur with av complete row of av, pv row on basal half, hind tibia with 3 av, 2 ad, 1 pd, without apical pv. Abdomen: Black, oviform in dorsal view, covered with yellow pruinosity, tergite 3 with black laddered patches, tergite 4 with black medial triangled patches, tergite 5 with black medial vittae, posterior margin of tergite 3, 4 and distal half of tergite 5 without patch, abdomen with thick long hairs, without spark patch, sternite 1 bare. Female: Forns two fifths of head width, or 1, fronto-orbital plates with small setae; both fore and mid tibia with 2ad, tergite 3-5 medial to both sides with wide and oblate triangled patches, other characters same as male. Etymology: The specific name refers to the type locality. Diagnosis: This new species is similar to P. paucispina Fang and Cui, 1988, but differs from it in male characters: the longest arista hairs 1.5 times as long as the arisa basal diameter (the longest arista hairs length half of postpedicel width in P. paucispina), gena about one fourth of eyes in height (gena about half of eyes in height in P. paucispina), veins brown (veins brownish in P. paucispina), halter fuscous (halter red yellow in P. paucispina), mid tibia without ad (mid tibia with 1 ad in P. paucispina), hind femur with pv row on basal half (hind femur with pv row completely in P. paucispina), hind tibia with 3 av (hind tibia with 2 av in P. paucispina), posterior of surstylus with hairs (posterior of surstylus without hairs in P. paucispina), surstylus protuberant backward distinctly (surstylus protuberant backward in P. paucispina).

Material examined: Holotype. 1♂ (IESNU), China, Yunnan Province, Mount Luquan (25.0458º N, 102.7100º E; 4100 m), 04.06.2000, Li Fuhua. 1♀ (IESNU), same data as holotype. Paratypes. 4♂♂ (IESNU), 1♀ (IESNU), same data as holotype.

Phaonia subpilipes Xue and Yu, sp. n. (Figs. 14-21) Male. Body length 6.5-7.3mm. 18 XUE, W., YU, T. Head: Eyes covered with long ciliae; frons about 2.5 times as wide as anterior ocellus, frontal vitta black, wider than fronto-orbital plate; 9-10 pairs of fr, expending to both sides of anterior ocellus, upper 4 pairs short and small, without ors; fronto-orbital plate, parafacial and gena with grey pruinosity; parafacial about 1.3 times as wide as flagellomere 1; antenna black, flagellomere 1 about 2.5 times as long as wide; arista long plumose, longest hairs about 1.3 times as long as flagellomere 1 wide; facial carina low, vibrissal angle situated in front of frontal angle in profile; anterior margin of gena with 2-3 rows of subvibrissal setae; gena about one third of eyes height, genal and postgenal hairs all black; proboscis short, prementum ruinose, about 2.5 times as long as wide; palpus black, subequal in length to prementum, labellum large.

15 16 17 14

18 19 20 21 Figs. 14-21. Phaonia subpilipes Xue and Yu, sp. n. (holotype). 14. Male abdomen (dorsal view). 15. Male sternite 5 (ventral view). 16. Male cerci (posterior view). 17. Male terminalia (profile view). 18. Female, spermatheca. 19. Female, sternites 1-5. 20. Female, ovipositor (dorsal view). 21. Female, ovipositor (ventral view). Scales: 14, 1.0 mm; 15, 0.5 mm; 16, 0.2 mm; 17, 0.2 mm; 18, 1.0 mm; 19, 1.0 mm; 20, 1.0 mm; 21, 1.0 mm. Thorax: Black in background color, covered with grey pruinosity; scutum with 4 black vittae; scutellum black; 0+1 acr, 2+3 dc, 0+2 ial, pra long and strong, about 2.5 times as long as posterior notopleural seta; notopleuron and katepimeron with hairs, lateral and lower margin of scutellum, basisternum of prosternum and meron bare; 1+2 katepisternal setae; anterior spiracles brown, posterior spiracles dark-brown. Wings: Slightly hyaline, veins brown, basicosta black; costal spine distinct; radial node entirely bare in dorsal and ventral view, r4+5 and m1+2 ­­straight; surroudings of r-m and m-m not clouded; calypters white to light-yellow and halteres yellow. Legs: Entirely black; fore tibia with 2 medium p; mid femur without distinct av, a row of long and large pv on basal half; mid tibia with 3 p, 2 pv; hind femur with complete 19 Review of the Phaonia consobrina Group from China av row, becoming long and strong apically, without pv; hind tibia with 6-7 av, complete ad row, distal part 2-3 setae slightly long and strong, with 1 ad in distal quarter, with 1-2 additional short subbasal ad, without apical pv; claws and pulvilli short and small, tarsi longer than tibiae. Abdomen: Black, oviform in dorsal view, covered with blue-grey pruinosity, without distinct shifting patch; tergite 3-5 with black medial vitta, both sides of vittae with large medial patch, sternite 1 bare. Female. Body length 6.5-7.5 mm. Frons about one third of head in width, frontal vitta about 4.0 times as wide as fronto-orbital plates, fr 5-6 pairs, ors 2 hypsokinesis, frontal triangle reaching one third lower of frons, parafacial about 1.3 times as wide as flagellomere 1; gena about two fifths of eye in height; katepimeron without hair or without hair on one side; fore tibia with 1 medium p, mid femur without av row, with thin and long pv on basal half, mid tibia with 1 pv; mid tibia with 4-5 av, 3-4 ad. Other main characters same as male. Etymology. The specific name refers to similar P. pilipes Ma and Feng, 1986. Diagnosis. This new species is similar to P. pilipes Ma and Feng, 1986, but it differs from the latter in having 3 posteriordc (4 posterior dc in P. pilipes); hind femur without pv (with a row of pv in P. pilipes); hind tibia with a row of ad (2 rows of ad in P. pilipes), abdomen medial vitta 1.5 times as wide as tibia diameter width (half of tibia diameter width in P. pilipes); free part of cerci with 2 pairs of long setae (free part of cerci with a pair of long setae in P. pilipes), anterior lateral margin of cerci distinct bend forward in lateral view (anterior lateral margin of cerci slightly straight in lateral view); the inner margin of cerci distinct projecting in lateral view (the inner margin of cerci indistinct projecting in lateral view).

Material examined: Holotype. 1♂ (IESNU), China, Mount Duoxiongla (29.4919º N, 94.9283º E; 3600-4200 m), Tibet, 3600-4200 m, 08.08.2003, Wang Mingfu. Paratypes. 24♂♂ (IESNU), 14♀♀ (IESNU), same data as holotype.

Species distributions

Phaonia angustinudiseta Xue, 1996

Material examined: Holotype. 1♂, China, Hejing, Xinjiang, 2350 m, 22.05.1960, Wang Shuyong. Paratypes. 2♂♂, same data as holotype; 1 ♂, Mt. Tianshan, Xinjiang, 02.05.1960, Wang Shuyong. Distribution: China: Xinjiang Uygur Autonomous Region (type locality: Hejing).

Phaonia cercoechinata Fang and Fan, 1986

Material examined: Holotype. 1♂, China, Mt. E’mei, Sichuan, 22.06.1984, Wu Jianyi, Paratypes. 1 ♂, Daocheng, Sichuan, 3950 m, 06.06.1982; 2 ♂♂, Xiangcheng, Sichuan, 3900-4000 m, 04.07.1982; 3 ♂♂, Mt. Moirigkawagarbo, Deqin, Yunnan, 4100 m, 29.07.1982; 1♂, Mt. Zhiben, yunlong, Yunnan, 3150 m, 23.06.1981. Distribution: China: Sichuan (type-locality: Mt. E’mei), Yunnan. 20 XUE, W., YU, T. Phaonia cercoechinatoida Feng and Ma, 2002

Material examined: Holotype. 1♂, China, Mt. Jiaoding, Hanyuan, Sichuan, 22.06.1987, Fengyan. Paratypes. 9♂♂, 3♀♀, same data as holotype; 3♂♂, Mt. E’mei, Sichuan, 02.06.1984, Fengyan; 4♂♂, Mt. Jiaoding, Sichuan, 26.06.1989, Li guangshi. Distribution: China: Sichuan (type-locality: Hanyuan, Mt. E’mei), Tibet Autonomous Region.

Phaonia dupliciseta Ma and Cui, 1992

Material examined: Holotype. 1♂, China, Gulian, Mohe, Heilongjiang, 01.06.1979. Cui Changyuan. Distribution: China: Heilongjiang (type-locality: Gulian).

Phaonia grunnicornis Xue, 1996

Material examined: Holotype. 1♂, China, Xinghai, Mt. E’la, Qinghai, 4500 m, 03.06.1964, Wang Shuyong. Distribution: China: Qinghai (type-locality: Mt. Ela).

Phaonia hesperia Feng, 2004

Material examined: Holotype. 1♂, China, Mt. Jiaoding, Shuanyuan, Sichuan, 3550 m, 11.07.1988, Fengyan. Paratypes. 2♂♂, same data as holotype; 1♂, Mt. Tuanbao, Hanyuan, Sichuan, 2800 m, 10.07.1988, Fengyan. Distribution: China: Sichuan (type-locality: Mt. Jiaoding).

Phaonia hirtirostris (Stein, 1907)

Material examined: Authors didn’t see specimens. (Hennig, 1964) Distribution: China: Ningxia, Sichuan, Tibet Autonomous Region (type-locality: Amdo); Russia, Uzbekistan, Kyrghyzstan.

Phaonia holcocerca Feng and Ma, 2000

Material examined: Holotype. 1♂, China, Mt. Erlang, Sichuan, 2790 m, 01.07.1988, Feng Lifu. Distribution: China: Sichuan (type-locality: Mt. Erlang), Yunnan.

Phaonia nigrinudiseta Xue and Zhang, 1996

Material examined: Holotype. 1♂, China, Mt. Geladandong, Qinghai, 5080, 13.06. 1990, Zhang Xuezhong. Distribution: China: Qinghai (type-locality: Mt. Geladandong).

Phaonia nudiseta (Stein, 1907)

Material examined: Authors didn’t see specimens. (Hennig, 1963) Distribution: China: Tibet Autonomous Region (type-locality: Amdo). 21 Review of the Phaonia consobrina Group from China Phaonia paucispina Feng and Cui, 1988

Material examined: Holotype. 1♂, China, Huzhong, Heilongjiang, 12.07.1978, Cui Changyuan. Distribution: China: Heilongjiang (type-locality: Huzhong).

Phaonia pilipes Ma and Feng, 1986

Material examined: Holotype. 1♂, China, Mt. Erlang, Sichuan, 3100 m, 11.07. 1983, Fengyan. Distribution: China: Sichuan (type-locality: Mt. Erlang).

Phaonia spuripilipes Fang and Fan, 1992

Material examined: Holotype. 1♂, China, Mt. Moirigkawagarbo, Deqin, Yunnan, 3350 m, 22.07.1982, Zhang Xuezhong. Distribution: China: Yunnan (type-locality: Mt. Moirigkawagarbo).

Phaonia subnudiseta Xue, 1996 Material examined: Holotype. 1♂, China, Batang, Yushu, Qinghai, 4600 m, 14. 06.1964, Wang Shuyong. Distribution: China: Qinghai (type-locality: Yushu, Batang).

Phaonia consobrina Zetterstedt, 1838

Material examined: Authors didn’t see specimens. (Zetterstedt, 1838) Distribution: Sweden (type-locality: Västerbotten, Lapland), Denmark, Russia.

Phaonia gergetica Zinovjev, 1994

Material examined: Authors didn’t see specimens. (Zinovjev, 1994) Distribution: Russia (type-locality: Caucasus), Georgia.

ACKNOWLEDGEMENTS This work was supported by the National Nature Science Foundation of China (No.31172139, No. 30870330), and the Foundation of Experimental Centre of Shenyang Normal University (SYZX200902). Special thanks are due to Li Fuhua, Wang Mingfu, Sun Hongkui and Zhang Xiang who collected the specimens.

REFERENCES Hennig, W., 1964, Muscidae. In: Linder, E. (Eds.). Die fliegen der palaearktischen Region 63b. Stuttgart, Schweizerbart, 772-899. Ma, Z. Y., Xue, W. Q., Feng, Y., 2002, Fauna Sinica Insecta. Diptera: Muscidae II, Phaoniinae I. Beijing, Science Press, 26: 421. McAlpine, J. F., 1981, Morphology and terminology-adults. In: McAlpine, J. F., Peterson, B. V., Shewell, G. E., Teskey, H. J., Vockeroth, J. R., Wood, D. M. (Eds.). Manual of Nearctic Diptera, Research, Agriculture Canada Research Branch 27, Ottawa, 1: 9-63. 22 XUE, W., YU, T.

Ringdahl, O., 1949, Forsök till artgruppering inem släktene Phaonia R.-D. och Helina R.-D. (fam. Muscidae). Entomologisk Tidskrift, 70: 136-146. Xue, W. Q., Zhang, C. T., Zhu, Y., 2006, Three new species of Phaonia R.-D. (Diptera: Muscidae) from China. Oriental Insects, 40: 137-134. Zinovjev, A. G., 1981, On the classification of Palaearctic species of the genus Phaonia R-D (Diptera: Muscidae). Entomologicheskoe Obozrenie, 60(3): 686-698.

Received: November 19, 2015 Accepted: June 01, 2017 J. Entomol. Res. Soc., 19(2): 23-30, 2017 ISSN:1302-0250

Pheromone Lures: Easy Way to Detect Trypodendron Species (Coleoptera: Curculionidae)

Jaroslav HOLUŠA Karolina LUKÁŠOVÁ*

Department of Forest Protection and Entomology, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague, Prague, CZECH REPUBLIC e-mail: *[email protected]

ABSTRACT Numbers of Trypodendron species captured with Trypowit® and Lineatin Kombi® lures were compared in 70 to 100 year old Norway spruce (Picea abies) stands at two localities (Kostelec and Tábor) in the Czech Republic. At each locality, five pairs of black, Theysohn window-slot traps were deployed; one trap of each pair contained Trypowit® lure and the other contained Lineatin Kombi® lure, and each trap of the pair was separated by 10 m. Among the Trypodendron species captured, T. lineatum was the most abundant, followed by T. domesticum and T. laeve. In most cases, the two kinds of lures caught similar numbers of beetles. T. lineatum males were more abundant than females at both localities in traps with Trypowit lures and in traps with Lineatin Kombi lures at Tábor. Males as well as females of T. domesticum were more abundant in traps with Lineatin Kombi lures than with Trypowit lures at Tábor but not at Kostelec. Both lures, which contain lineatin and attract all Trypodendron species including T. laeve, can be used to monitor the abundance of these bark beetle species. The Lineatin Kombi lure, however, contains more alcohol and is slightly more effective than the Trypowit lure for monitoring Trypodendron species on broadleaf trees. Key words: Bark beetles, central Europe, flight activity, pheromones, sex ratio.

INTRODUCTION Of the four Trypodendron species that occur in Central Europe, two prefer coniferous trees and two prefer broadleaf trees. The striped ambrosia beetle, Trypodendron lineatum (Olivier, 1795), is a serious pest of conifers in the Palearctic region and in North America (Wood, 1982). Trypodendron laeve Eggers, 1939 is widespread in Europe and also attacks conifers (Muona, 1994; Martikainen et al., 1996; 1999; Holzschuh, 1990a, 1990b, 1995; Krehan and Holzschuh, 1999; Martikainen, 2000; DAISIE, 2009; Kirkendall and Faccoli, 2010; Lukášová et al., 2012; Lukášová and Holuša, 2014). The European hardwood ambrosia beetle (Trypodendron domesticum (Linnaeus, 1758)) is a pest of broadleaf, hardwood trees (Schwenke, 1974; Schwerdtfeger, 1981). Trypodendron signatum (Fabricius, 1787) occurs in oak forests in lowland and hills and is generally the least abundant of the four species (Pfeffer, 1989; Lukášová et al., 2012) but can be very abundant (Gaubicher et al., 2003). The most important method for control of Trypodendron species is the logging and removal of all stored wood from forests before T. lineatum swarms each year. 24 HOLUŠA, J., LUKÁŠOVÁ, K. Trypodendron species can not infest wood from the end of May until February of the following year because the species are inactive at this time. Wood to be harvested should not be left in areas where Trypodendron species were abundant in the previous year, because the beetles will overwinter in the litter or wood of such trees. From a practical perspective, it is not necessary to distinguish wood attacked by T. lineatum and T. laeve, because the two species are very similar in terms of bionomics, damage, and control (Bussler and Schmidt, 2008). Lures containing lineatin (the pheromone produced by Trypodendron females) are useful for monitoring the abundance of all Trypodendron species but are probably ineffective for mass trapping. The population density of T. lineatum, for example, was not be substantially influenced by continuous mass trapping with lineatin-baited traps (Dimitri et al., 1992). A cost/benefit analysis also indicated that the cost of mass trapping exceeded the benefit (König, 1992). Still, pheromone lures could be used for determining whether a site is suitable for storing wood. If more than 500 T. lineatum beetles are captured during the flight period, the risk of stored wood infestation is very high (Zahradník, 2002). The use of pheromone-based lures to attract Trypodendron species facilitates the determination of dates when swarming by these pests begins and ends. Data from traps containing pheromone-based lures enable foresters to take early preventive measures that will reduce the damage resulting from bark beetle infestation. It is also much easier to determine which species are present by examining specimens in traps containing pheromone than by cutting specimens from wood or by capturing specimens that emerge from wood in emergence traps. Pheromone lures for T. lineatum have been used for a relatively long time, and all of them contain lineatin and also other substances in some cases (Klimetzek et al., 1981; Schurig et al., 1982; King et al., 1983; Lukášová and Holuša, 2014). New lures that are attractive for other Trypodendron species have been produced only recently. The Austrian company Witasek Pflanzenschutz GmBH provides the pheromone lure Trypowit® for capturing T. lineatum and the pheromone lure Lineatin Kombi® for capturing other Trypodendron species including T. domesticum and T. signatum (http://www.witasek.com/). The Lineatin Kombi lure was developed at the CHEMIPAN Research and Development Laboratories (www.sciencedaily.com/ releases/2011/09/110915083651.htm). Field tests with the Lineatin Kombi lure have been carried out by researchers from the Technical University of Dresden in forests near Leipzig, Dresden, and Wermsdorf. The forests consisted of common beeches, oaks, and ashes. The researchers reported that high numbers of T. domesticum and T. lineatum were captured in the pheromone traps; T. signatum specimens were also trapped (www.sciencedaily.com/releases/2011/09/110915083651.htm). The main aim of study was to compare numbers of Trypodendron species captured in traps containing Trypowit® and Lineatin Kombi® lures in spruce forests in the Czech Republic. A second aim was to determine whether the Lineatin Kombi lure attracts T. laeve, which was recently detected the Czech Republic, Poland, and Romania (Lukášová et al., 2012; Olenici et al., 2014). 25 Pheromone lures: Easy Way to Detect Trypodendron Species MATERIALS AND METHODS Trapping experiments were conducted in 70 to 100 year old Norway spruce (Picea abies Karsten) stands at two localities in the Czech Republic (Kostelec nad Černými lesy; 49°58’N, 14°49’E and Tábor; 49°31’N 14°33’E) from March to July 2014. At each locality, five pairs of pheromone-baited, black window-slot traps (Theysohn, Germany) were deployed. Each trap had a total active surface area of 4,284 cm2 (42 x 51 cm on two sides). The traps in each pair were 10 m apart, 10-15 m from the nearest forest edge, and 1.5-2.0 m above the soil surface. Adjacent pairs of traps at each locality were 50-100 m apart. Each pair of trap was baited with a standard synthetic pheromone lure. One trap of the pair was baited with Trypowit® and the other with Lineatin Kombi® (http:// www.witasek.com/). The main active chemicals in the Trypowit lure are alfa-pinen (2,6,6-trimethyl-bicyclo-[3,1,1]hept-3-en) and lineatin (3,3,7-trimethyl-2,9-diox- atricyclo-[3.3.10 4,7] nonane). The main active chemicals in the Lineatin Kombi lure are lineatin (3,3,7-trimethyl-2, 9-dioxatricyclo-[3.3.10 4,7] nonane), qublaiacol (2-methoxyphenol), nonyl aldehyde, and 3-hydroxy-2-methyl-2-butanone. Once either kind of lure is removed from its hermetically sealed, three layered aluminium-plastic laminated pouch, the aggregation pheromone contained in a cellulose plate is gradually released through a permeable polyethylene film. The traps with aggregation pheromone were deployed in mid-March of 2014 and were monitored until the time when two consecutive collections were negative, which occurred in early June/July 2014. Beetles were collected every 7 days and were stored in 2.5-ml Eppendorf microtubes at <−5°C. Individual beetles were identified to species according to Pfeffer (1989) and Bussler and Schmidt (2008). Sex was determined according to secondary sex characteristics (Pfeffer, 1989). Because the data were not normally distributed (as indicated by the Shapiro Wilk test), the number of beetles per trap in traps with the two kinds of lures was compared using Wilcoxon signed-rank test, and the number of beetles per trap across lure types was compared between the two localities using a Mann-Whitney U Test. All tests were performed with STATISTICA 12.0 software.

RESULTS Data concerning the numbers of Trypodendron species trapped as affected by lure type, beetle species, beetle sex and locality are presented in Table 1. A total of 10, 822 beetles were captured and three species of Trypodendron were recorded. T. lineatum was the most abundant (n=8,451), followed by T. domesticum (n=2,238) and T. laeve (n=133). Numbers were more than several times higher for T. lineatum (Z=3.43; p<0.001) and T. laeve (Z=3.78; p<0.001) at Kostelec than at Tábor. For T. domesticum, numbers were only slightly higher at Kostelec than at Tábor (Z=2.38; p<0.05). 26 HOLUŠA, J., LUKÁŠOVÁ, K. For T. lineatum, males were more abundant than females in traps with the Trypowit lure at both localities and in traps with the Lineatin Kombi lure at Tábor. Males as well as females of T. domesticum were more abundant with the Lineatin Kombi lure than with the Trypowit lure at Tábor. Averaged across all Trypodendron species, the numbers of males and females captured were usually similar with both kinds of lures but were significantly different in several cases. Males of T. lineatum were slightly but significantly more abundant with the Trypowit lure than with the Lineatin Kombi lure at Kostelec. Males as well as females of T. domesticum were slightly but significantly more abundant with the Lineatin Kombi lure than with the Trypowit lure at Tábor (Table 1).

DISCUSSION In the current study, the most abundant species of Trypodendron was T. lineatum, followed by T. domesticum and T. laeve. The results confirm earlier observations that Trypodendron species are attracted by lineatin (Klimetzek et al., 1981; Schurig et al., 1982; King et al., 1983; Paiva and Kiesel, 1985; Kvamme, 1986; Krehan and Holzschuh, 1999; Martikainen, 2000; Lukášová et al., 2012; Holuša and Lukášová, 2014). The failure to trap T. signatum, which also occurs in the Czech Republic, can be explained by the low numbers of its host trees in the study areas (Lukášová et al., 2012). The pheromone traps in the current study were located in Norway spruce forests, and Norway spruce is not a host of T. signatum. We tested two lures in spruce areas with different population densities of Trypondendron species T. lineatum has been abundant in forests surrounding the town of Kostelec nad Černými lesy for a long time. The numbers of T. lineatum caught by the different lures were similar in most cases. Males but not females ofT. lineatum were more abundant in traps containing the Trypowit lure rather than the Lineatin Kombi lure at Kostelec, while both males and females of T. domesticum were more abundant in traps containing the Lineatin Kombi lure rather than the Trypowit lure at Tábor. That both lures generally attracted similar numbers Trypondendron species can be explained by the fact that both lures contain lineatin. In addition to lineatin, the Trypowit lure also contains α-pinen, which attracts T. lineatum but repels the two other Trypondendron species that attack broadleaf trees (Paiva, 1982; Paiva and Kiesel, 1985). Lures that included α-pinene with the pheromone, either with or without ethanol, attracted significantly moreT. lineatum than those with the pheromone alone (Shore and Lindgren, 1996). Exposure of T. lineatum males to ethanol increased their frequency of steady, upwind flight; however, only lineatin was effective in inducing them to land on and enter traps (Salom and McLean, 1990). Ethanol at a release rate of 1 g/day inhibited the response of North American T. lineatum to lineatin, whereas ethanol at 310 and 500 mg/day acted synergistically with lineatin for European T. lineatum. α-pinene was synergistic with lineatin for the capture of European T. lineatum in traps that simulated tree trunks (Borden et al., 1982). 27 Pheromone lures: Easy Way to Detect Trypodendron Species

Table 1. Numbers Trypodendron lineatum, T. domesticum, and T. laeve males and females caught in traps and sex ratios as affected by lure type.

Statistical comparisonsb Numbers of beetles Locality Species Sex Lure per trap (mean±SE)a Effect of lure type Sex ratio in traps Sex ratio in on numbers of with Lineatin traps with Try- beetles per trap1 Kombi lures2 powit lures3 Lineatin T. lineatum Male 266.6±61.2 Kombi 2.02 * T. lineatum Male Trypowit 905.6±256.9 2.02 * 2.02 * Lineatin T. lineatum Female 166.2±39.2 Kombi 1.21 ns T. lineatum Female Trypowit 214.2±53.3

Lineatin T. domesticum Male 67.4±17.6 Kombi 0.94 ns T. domesticum Male Trypowit 91.8±29.7 Kostelec 0.27 ns 1.46 ns Lineatin T. domesticum Female 67.8±15.1 Kombi 0.67 ns T. domesticum Female Trypowit 78.4±25.0

Lineatin T. laeve Male 6.2±1.9 Kombi 0.53 ns T. laeve Male Trypowit 8.4±4.0 0.18 ns 1.21 ns Lineatin T. laeve Female 5.4±2.3 Kombi 0.26 ns T. laeve Female Trypowit 5.8±2.7

Lineatin T. lineatum Male 34.0±14.2 Kombi 0.67 ns T. lineatum Male Trypowit 50.2±26.3 0.67 ns 2.02 * Lineatin T. lineatum Female 31.2±12.8 Kombi 0.67 ns T. lineatum Female Trypowit 22.2±13.6

Lineatin T. domesticum Male 66.8±27.6 Kombi 2.02 * T. domesticum Male Trypowit 21.6±8.4 Tábor 2.02 * 2.02 * Lineatin T. domesticum Female 44.8±19.2 Kombi 2.02 * T. domesticum Female Trypowit 9.0±3.9

Lineatin T. laeve Male - Kombi - T. laeve Male Trypowit - - 1.60 ns Lineatin T. laeve Female - Kombi 1.60 ns T. laeve Female Trypowit 0.8±0.4 a Across the entire sampling period in 2014. bns = nonsignificant, and * = significant at p<0.05 according to Wilcoxon signed-rank tests. For sex ratio, a significant effect indicates that the ratio was different from 1:1). 1Number of beetles per trap with the two types of lures comparing using Wilcoxon signed-rank test, 2Comparison of males and females abundance per trap with Lineatin Kombi lures using Wilcoxon signed- rank test, 3Comparison of males and females abundance per trap with Trypowit lures using Wilcoxon signed-rank test. 28 HOLUŠA, J., LUKÁŠOVÁ, K. In addition to lineatin, the Lineatin Kombi lure contains quaiacol, nonyl aldehyde, and 3-hydroxy-2-methyl-2-butanone. These other substances are added because isomers of 2-methoxy-phenol and methyl-butanol can attract T. domesticum (Holighaus and Schütz, 2006). The Lineatin Kombi lure does not contain ethanol, although both T. domesticum (Petercord, 2006) and T. signatum (Galko et al., 2013) are attracted by lures that contain ethanol. The producer’s literature for the Trypowit and Lineatin Kombi lures (http://www. witasek.com/) does not refer to T. laeve, and the pheromones of T. laeve have not been studied. T. laeve was previously caught in traps with lures containing lineatin in alcohol (Martikainen, 2000; Grégoire et al., 2001; Lukášová and Holuša, 2014). The sex ratio of T. lineatum was significantly shifted toward males in three of four cases (two lures × two localities) while the sex ratio of T. domesticum was significantly shifted toward males with both kinds of lures at the Tábor locality but with neither kind of lure at the Kostelec locality. Pheromone traps should catch more males than females because the trapped females produce additional sex pheromones that attract males (Schwenke, 1974; Schwerdtfeger, 1981). In a previous study using lures containing lineatin and ethanol or α-pinene, the sex ratio of captured bark beetles was more male biased for T. domesticum than for T. lineatum or T. signatum (Paiva, 1982). The proportion of females trapped depends on ethanol and/or α-pinene, i.e., significantly more female beetles were trapped when lures contained linatin plus ethanol and/or α-pinene rather than lineatin alone (Shore and Lindgren, 1996). That both Trypowit and Lineatin Kombi lures (http://www.witasek.com/) contain lineatin probably explains why the lures attracted all species of Trypodendron in the two study localities (T. lineatum, T. domesticum, and T. laeve) and why differences in the numbers trapped were not greatly affected by kind of lure. The Lineatin Kombi lure may attract more T. domesticum than the Trypowit lure because the Lineatin Kombi lure also contains alcohols. This indicates that the Lineatin Kombi lure may be especially useful for monitoring Trypodendron species on broadleaf trees. The sex ratio of Trypodendron species in traps containing Trypowit and Lineatin Kombi lures were male biased probably because the lures contain lineatin, which is a female sex pheromone that attracts males. The results are consistent with the view that T. laeve is widespread throughout Europe but is less abundant than T. lineatum.

ACKNOWLEDGEMENT This research was supported by project QJ1330233 of the Ministry of Agriculture of the Czech Republic and IGA B05/16 of the Czech University of Life Sciences. The authors thank Gale A. Kirking for linguistic and editorial improvements. The authors also thank Jana Dandová and Bára Vostřáková for help with field work. 29 Pheromone lures: Easy Way to Detect Trypodendron Species REFERENCES

Borden, J. H., King, C. J., Lindgren, S., Chong, L., Gray, D. R., Oehlschlager, A. C., Slessor, K. N., Pierce, H. D. Jr., 1982, Variation in response of Trypodendron lineatum from two continents to semiochemicals and trap form. Environmental Entomology, 1: 403-408. Bussler, H., Schmidt, O., 2008, Trypodendron laeve Eggers, 1939 - Ein wenig bekannter Nutzholzborken- käfer. Forstschutz Aktuell, Wien, 45: 11-13. DAISIE, 2009, Handbook of alien species in Europe. Springer, Netherlands: Dordrecht, 399. Dimitri, L., Gebauer, U., Losekrug, R., Vaupel, O., 1992, Influence of mass trapping on the population-dynamic and damage-effect of bark beetles.Journal of Applied Entomology, 114: 103-109. Galko, J., Nikolov, C., Gubka, A., Vakula, J., 2013, Účinnosť odparníkov na báze etanolu na drevokazných škodcov v dubových porastoch. (Efficiency of lures based on ethanol to wood decaying pestsin oak stands). In: Kunca, A. (Ed.). Aktuálne problémy v ochrane lesa 2013, Zborník referátov z 22. medzinárodnej konferencie, 25 - 26 April 2013; Nový Smokovec, Slovakia, NLC, Zvolen, 137-141. Gaubicher, B., De Proft, M., Grégoire, J.-C., 2003, Trypodendron domesticum and Trypodendron signatum: two scolytid species involved in beech decline in Belgium. In: McManus, M. L., Liebhold, A. M. (Eds.). Proceedings: Ecology, Survey and Management of Forest Insects, 1-5 September 2002; Krakow, Poland: Gen. Tech. Rep. NE-311, Newtown Square, PA: U.S. Department of Agriculture, Forest Service, Northeastern Research Station, 134-135. Grégoire, J.-C., Piel, F., De Proft, M., Gilbert, M., 2001, Spatial distribution of ambrosia-beetle catches: a possibly useful knowledge to improve mass-trapping. Integrated Pest Management Reviews, 6: 237-242. Holighaus, G., Schütz, S., 2006, Odours of wood decay as semiochemicals for Trypodendron domesticum L. (Col., Scolytidae). Mitteilungen der Deutschen Gesellschaft für Allgemeine und Angewandte Entomologie, 15: 161-165. Holzschuh, C., 1990a, Ein neuer, gefährlicher Nutzholzborkenkäfer in Österreich. Forstschutz Aktuell, Wien, 3: 2. Holzschuh, C., 1990b, Ergebnisse von Untersuchungen über die Einschleppung von Borkenkäfern an Holzlager- und Umschlagplätzen. Forstschutz Aktuell, Wien, 5: 7-8. Holzschuh, C., 1995, Forstschädlinge, die in den letzten fünfzig Jahren in Österreich eingewandert sind oder eingeschleppt wurden. Stapfia, 37: 129-141. King, C. J., Oehlschlager, A. C., Borden, J. H., 1983, Response of Trypodendron lineatum (Olivier) to isomers of its aggregation pheromone, lineatin, in England. Journal of Applied Entomology, 95: 531-533. Kirkendall, L. R., Faccoli, M., 2010, Bark beetles and pinhole borers (Curculionidae, Scolytinae, Platypodinae) alien to Europe. ZooKeys, 56: 227-251. Klimetzek, D. K., Kiesel, K., Möhring, C., Bakke, A., 1981, Trypodendron lineatum: reduction of pheromone response by male beetles. Naturwissenschaften, 68: 149-150. König, E., 1992, Mass trapping of Trypodendron lineatum Ol (Col, Scolytidae) - impact on the infestation of stored logs and cost-benefit-analysis of a 5 year field experiment. Journal of Applied Entomology, 114: 233-239. Krehan, H., Holzschuh, C., 1999, Trypodendron laeve - Vorkommen in Österreich. Forstschutz Aktuell, Wien, 23/24: 6-8. Kvamme, T., 1986, Trypodendron piceum Strand (Col.: Scolytidae): Flight period and response to synthetic pheromones. Fauna Norvegica, Serie B, 33: 65-70. Lukášová, K., Holuša, J., 2014, Comparison of Trypodendron lineatum, T. domesticum and T. laeve (Coleoptera: Curculionidae) flight activity in Central Europe. Journal of Forest Science, 60: 382-387. Lukášová, K., Knížek, M., Holuša, J., Čejka, M., Kacprzyk, M., 2012, Is the bark beetle Trypodendron laeve (Coleoptera: Curculionidae: Scolytinae) an alien pest in the Czech Republic and Poland? Polish Journal of Ecology, 60: 789-795. 30 HOLUŠA, J., LUKÁŠOVÁ, K.

Martikainen, P., 2000, Flight period and ecology of Trypodendron proximum (Niijima) (Col., Scolytidae) in Finland. Journal of Applied Entomology, 124: 57-62. Martikainen, P., Siitonen, J., Kaila, L., Punttila, P., 1996, Intensity of forest management and bark beetles in non-epidemic conditions: a comparison between Finnish and Russian Karelia. Journal of Applied Entomology, 120: 257-264. Martikainen, P., Siitonen, J., Kaila, L., Punttila, P., Rauh, J., 1999, Bark beetles (Coleoptera, Scolytidae) and associated beetle species in mature managed and old growth boreal forests in southern Finland. Forest Ecology and Management, 116: 233-245. Muona, J., 1994, Tarkennuksia eraiden kuoriaislajien esiintymiseen Suomessa ja Venajan Karjalassa (Coleoptera). [Revisions to the occurrence of some beetle species in Finland and Russian Karelia (Coleoptera).] Sahlbergia, 1: 7-10. Olenici, N., Knížek, M., Olenici, V., Duduman, M.-L., Biriş, I.-A., 2014, First report of three scolytid species (Coleoptera: Curculionidae, Scolytinae) in Romania. Annals of Forest Research, 57: 89-97. Paiva, M. R., 1982, Interference among pheromone traps for the ambrosia beetles Trypodendron spp. Journal of Applied Entomology, 94: 180-186. Paiva, M. R., Kiesel, K. 1985, Field responses of Trypodendron spp. (Col., Scolytidae) to different concentrations of lineatin and α-pinene. Journal of Applied Entomology, 99: 442-448. Petercord, R. 2006, Flight period of the broad-leaved ambrosia beetle Trypodendron domesticum L. in Luxembourg and Rhineland-Palatinate between 2002 and 2005. IUFRO Working Party 7.03.10. Proceedings of the Workshop 2006, Gmunden, Austria, 213-218. Pfeffer, A., 1989, Kůrovcovití Scolytidae a jádrohlodovití Platypodidae. [Bark beetles Scolytidae and ambrosia beetles Platypodidae.]. Academia, Prague, Czech Republic, 137pp. Salom, S. M., McLean, J. A., 1990, Flight and landing behavior of Trypodendron lineatum (Coleoptera: Scolytidae) in response to different semiochemicals. Journal of Chemical Ecology, 16: 2589-2604. Shore, T. L., Lindgren, B. S., 1996, Effect of ethanol and α-pinene on response of ambrosia beetle, Trypodendron lineatum, to lineatin-baited funnel and drainpipe traps. Journal of Chemical Ecology, 22: 2187-2196. Schurig, V., Weber, R., Klimetzek, D., Kohnle, U., Mori, K., 1982, Enantiomeric composition of ‘Lineatin' in three sympatric ambrosia beetles. Naturwissenschaften, 69: 602-603. Schwenke, W., 1974, Die Forstschädlinge Europas. Käfer. Verlag Paul Parey, Hamburg, Germany. Schwerdtfeger, F., 1981, Waldkrankheiten. Paul Parey, München, Germany. Wood, S. L., 1982, The bark and ambrosia beetles of North and Central America (Coleoptera: Scolytidae), a taxonomic monograph. Great Basin Naturalist Memoirs, 6: 1-1359. Zahradník, P., 2002, Dřevokaz čárkovaný Trypodendron (=Xyloterus) lineatum (Ol.). [Striped ambrosia beetle Trypodendron (=Xyloterus) lineatum (Ol.).] Lesnická práce, 8(Supplementum): I-IV.

Received: September 25, 2015 Accepted: June 15, 2017 J. Entomol. Res. Soc., 19(2): 31-42, 2017 ISSN:1302-0250

Alien Species Numerically Dominate Natural Enemy Communities in Urban Habitats: A Preliminary Study

Leandro Sebastian WAGNER* María Silvina FENOGLIO Adriana SALVO

Instituto Multidisciplinario de Biología Vegetal (IMBIV)-CONICET, Centro de Investigaciones Entomológicas de Córdoba (CIEC)-Facultad de Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, (X5016GCA), Córdoba, REPÚBLICA ARGENTINA e-mails: *[email protected], [email protected], [email protected]

ABSTRACT Urbanization generally leads to reduced biodiversity of insects, mainly native species. Knowing the structure of natural enemy communities is important because of the significant role they play as biological control agents of herbivores. Here, we evaluated the influence of urbanization on the complex of natural enemies of the aphid Aphis gossypii (Hemiptera Aphididae), which feeds on the flowers of jacaranda (Jacaranda mimosifolia, Bignoniaceae). Seven sites with different urbanization levels were selected in the city of Córdoba (central Argentina) and three jacaranda trees with aphids were sampled in each site. Diversity, a and relative contribution of native and alien species were estimated. In general, the results indicate that urbanization did not have a significant effect on the community of natural enemies, with species richness showing a marginally significant increase with increasing urbanization levels. Although a similar richness of native and alien species was recorded across sites, 80% of natural enemy abundance belonged to alien species. Aphid populations were found to increase with increasing level of urbanization, but this variable did not have an influence on natural enemies. The dominance of alien and/or generalist species resistant to disturbances might account for the lack of relationship between urbanization and structure of natural enemy communities. Key words: Aphids, parasitoids, predators, hyperparasitoids, urbanization.

INTRODUCTION Urban environments are complex heterogeneous and disturbed systems, characterized by reduced and isolated natural habitats, high pollution levels, elevated temperatures, compacted soils and great amounts of concrete, among other features (Pickett et al., 2011; Jones and Leather, 2012). In general, these conditions have negative effects on different groups of terrestrial animals (McKinney, 2008), including insects (Kotze et al., 2011; Jones and Leather, 2012), causing declines in abundance and species richness and producing changes in community structure, such as increased dominance of alien (McKinney, 2002; McDonald et al., 2013), opportunistic and generalist species (Faeth et al., 2005). Alien species sometimes replace native ones faster than they are lost (McKinney, 2008); hence, urbanization may favor an increase in biodiversity at the local scale (Frankie and Ehler, 1978; Niemelä, 1999). The process of replacement of local biotas by nonindigenous and locally expanding 32 WAGNER, L. S., FENOGLIO, M. S., SALVO, A. species that can co-exist with humans is known as biotic homogenization (McKinney and Lockwood, 1999). This process promotes the geographical expansion of some species, the “winners”, and the geographical reduction of others, the “losers” (Baskin, 1998). Natural enemies, predators and parasitoids, play a fundamental role in the control of insect herbivores in urban habitats (Losey and Vaughan, 2006; Kotze et al., 2011). However, little is known about the effects of urbanization on parasitoids (reviewed in Fenoglio and Salvo, 2010) as well as on predatory species belonging to families other than Carabidae (Denys and Schmidt, 1998; Zanette et al., 2005; Ferrante et al., 2014) and, to date, there is no evidence of the impact of urbanization on secondary parasitoids (hyperparasitoids). Natural enemies, belonging to higher levels of the food chains, are particularly sensitive to habitat fragmentation (Hunter, 2002), even to that resulting from urbanization. Primary parasitoids may be negatively affected by urbanization or not affected at all (reviewed in Fenoglio and Salvo, 2010), whereas predatory carabid beetles might exhibit declines in species richness with increasing urbanization (Martinson and Raupp, 2013). In the present research we assessed the effects of urbanization on the complex of natural enemies (predators, parasitoids and hyperparasitoids) of the aphid Aphis gossypii Glover, 1877 (Hemiptera Aphididae) present on Jacaranda mimosifolia D. Don, 1822 (Bignoniaceae) trees in Córdoba city, Argentina. Many aphid species are considered pests of crops and ornamental plants and their abundance, as in other herbivores, often increases with increasing levels of urbanization (Raupp et al., 2010). Among their natural enemies, the alien species ladybird Harmonia axyridis (Pallas) has shown great potential as a biological control agent of A. gossypii (Flores Macías et al., 2010). However, H. axyridis has been categorized as an invasive alien species in different parts of the world because it displaces native species (Martins et al., 2009; Roy et al., 2012), which may be happening also in Argentina (Saini, 2004). Therefore, studying the community structure of natural enemies of aphids in cities is important in order to develop managing strategies of pest herbivores but also for conservation of beneficial native insects. Here we evaluated α-diversity of natural enemies and the proportion of native and alien species, and examined possible differences in species composition (β-diversity) in relation to the urbanization level in a city. To discard possible indirect effects of urbanization (mediated through changes in herbivore abundance) on natural enemy communities (Koricheva, 1994), we also estimated the effect of urbanization level on aphid abundance and, therefore, its impact on the third trophic level. We expect a higher a-diversity of natural enemies in sites with lower urbanization due to a higher dominance of native species. In addition, we expect a decrease in b-diversity at higher levels of urbanization mainly due to a higher presence of few alien species resistant to environmental disturbances.

MATERIALS AND METHODS

Study area The present study was conducted in the city of Córdoba, located in central Argentina (31° 20’ S 64° 10’ W, 440 masl). The city covers an area of 562 km² (Dirección de 33 Alien Species Numerically Dominate Natural Enemy Communities Catastro Córdoba, 2007) and has a population of 1,330,023 inhabitants (INDEC, 2010). Seven sites with different urbanization indices, located along a 10-km northwest to southeast transect, were selected from 18 sites previously used by Fenoglio et al. (2009). The selection of sites was based on two criteria: 1) that represented an effective gradient of urbanization based on the index (Fig. 1), 2) that jacaranda trees were infected with aphids at the period of the sampling. The urbanization index corresponds to the inverse of the first ordination axis of a Principal Component Analysis (PCA) based on several urbanization variables (distance from the city core, vehicular traffic, garden surface, vegetation coverage, ground temperature), which accumulated 74% of the variance. The index corresponding to the sites here used varied from -1.84 (least urbanized) to 4.16 (most urbanized).

Fig. 1. Location of sampling sites arranged in increasing order of urbanization (sites 1 to 7) in Córdoba city (Argentina).

Study system The host plant J. mimosifolia, commonly known as “jacaranda”, is a subtropical tree widely present in cities, with deciduous foliage and large blue-violet flowers, grouped in panicles, which bloom in October in the study area (Dimitri, 1980). The aphid A. gossypii is a polyphagous herbivore with cosmopolitan distribution (Delfino et al., 2007; Flores Macías et al., 2010), which attacks crops and ornamental plants (Margaritopoulos et al., 2006). In Córdoba city, it is associated with jacaranda trees, among several other plant species (Delfino and Buffa, 2008). Aphids have a varied fauna of natural enemies, comprising predators (Coleoptera, Neuroptera and Diptera) and primary parasitoids and hyperparasitoids (Hymenoptera).

Sampling During October and November 2012, three jacaranda trees attacked by A. gossypii were selected at each site, with the exception of two sites where only one and two 34 WAGNER, L. S., FENOGLIO, M. S., SALVO, A. jacaranda trees with aphids were found. The selected trees were similar on their age and architecture (estimated as trunk diameter at breast height, ranging from 100 to130 cm, and a height between 3-5 m), and no chemical treatment was applied to them. In cases where more than three attacked trees were observed, focal trees were selected based upon their foliage accessibility (canopy height not longer than the length of the pruning scissor which was of 7 m), and separated from each other by a distance equal to or greater than 100 m. In each tree, 20 inflorescences of about 30 cm in length were selected at random around the outline of trees, located between 1 and 7 m in height, to account for the abundance of aphids and their natural enemies. Aphid colonies are aggregated; hence, to facilitate quantification ofA. gossypii abundance, an index proposed by Yokomi and Tang, (1996) was used. Thus, aphid samples were classified as follows: 0 = no aphids; 1 = 1-20 aphids; 2 = 21-50 aphids; 3 = 51-100 aphids; 4 = 101-200 aphids; 5 => 200 aphids. Then, the abundance of aphids was estimated in each sample using the midpoints of the above categories: 0, 10, 35, 75, 150 and 300, respectively, following Yokomi and Tang (1996). To determine species richness and abundance of natural enemies, other five inflorescences of about 30 cm in length were randomly taken, placed in labeled plastic bags and transported to the laboratory where material was bred. The inflorescences were placed in a vase containing water to keep them hydrated, all inside a plastic container of 30 cm in height and 30 cm in diameter covered with a plastic mesh to allow proper ventilation. The immature stages of natural enemies were maintained at 18°C -22°C temperature, 55%- 68% R.H., and 12L:12D photoperiod, and were fed daily with A. gossypii individuals, until they reached the adult stage. Adults were placed in tubes with 70% ethanol. Species were identified using dichotomous keys (Gonzalez 2006; Zumoffenet al., 2015) and the assessment of a specialist (Dr. Leticia Zumoffen, INTA Rafaela). The origin and the feeding habit of natural enemies were obtained from literature (Weems 2004; Gonzalez 2006; López et al., 2012) and the assessment of a specialist (Dr. Leticia Zumoffen, INTA Rafaela). The specimens were deposited in the Collection of Entomology Department, National University of Córdoba, Argentina. The following variables related to natural enemies were estimated for each site: α-diversity measured through species richness (total number of species found in the period sampled), abundance (total number of individuals found in the period sampled) and Shannon index; relative richness of native species; relative abundance of native species and β-diversity estimated by the quantitative Sorensen index. The δ-diversity was measured as the total number of natural enemies found throughout the study sites.

Statistical analyses The effect of urbanization on the natural enemies of the aphid A. gossypii was evaluated using simple linear regression analyses, considering the index of urbanization as the independent variable and a-diversity, relative richness and abundance of native species as dependent variables. Richness was estimated using the rarefaction method with the package vegan in R 2.9.2 statistical software (R Core Team, 2010), which enables the comparison of samples with different numbers 35 Alien Species Numerically Dominate Natural Enemy Communities of individuals (Magurran, 2004). Moreover, to discard possible indirect effects of urbanization on natural enemies due to changes in resource availability, we first examined whether aphid abundance varied according to the urban gradient and then if/how resource abundance influenced indicators of diversity. It was not possible to perform multiple linear regression analysis due to the limited number of degrees of freedom available and multicollinearity problems (Graham, 2003). In all cases, a was of 0.05, although probability values ranging between 0.05 and 0.1 were considered marginally significant. The effect of urbanization on the composition of natural enemies in different sites was evaluated using a partial Mantel test (Legendre and Legendre, 1998) between the distance matrix of Sorensen, expressed as the additive inverse (1 - Sorensen index) and the Euclidean matrix of urbanization distances (expressed as the differences between sites in terms of their urbanization index), controlling for geographical distances between sites (expressed in km). This analysis was performed using the vegan package in R software.

RESULTS The 90 jacaranda inflorescences sampled held 230 individuals corresponding to 14 species of natural enemies (δ-diversity) associated with the aphid A. gossypii (Table 1). Predators made the greatest contribution (72%) to the species richness of natural enemies. In terms of abundance, primary parasitoids and hyperparasitoids together were the group most abundant with 66% of all natural enemies (Table 1). Among the predators, H. axyridis was the dominant species. The species richness of natural enemies associated with A. gossypii showed a slight increasing trend at sites with high urbanization level (Table 2 and Fig. 2a). The same trend was observed for the richness corrected by rarefaction (Table 2). On the other hand, neither abundance nor Shannon index of natural enemy community changed with the urban gradient (Table 2). Although abundance of aphids significantly increased as the level of urbanization increased (Table 2 and Fig. 2b), none of the variables indicative of a-diversity were related to aphid abundance (Table 2). In terms of richness, a similar proportion of native and alien species associated with A. gossypii was recorded (Table 1 and Fig. 3); however, when considering the abundance, more than 80% of individuals belong to alien species (Fig. 3). Moreover, most of these alien species were present in a high proportion of the studied sites (Table 1). Both relative richness and abundance of native natural enemies per site were less than 41% and showed no significant changes in relation to the urbanization index or the abundance of aphids (Table 2). The Partial Mantel test showed that species composition of natural enemies evaluated through β-diversity (Sorensen distances) was independent of the level of urbanization (rM = 0.22, p = 0.19). 36 WAGNER, L. S., FENOGLIO, M. S., SALVO, A. 0.14 1.00 0.86 0.86 0.14 0.57 0.14 0.57 0.29 0.14 0.71 0.57 0.71 1.00 species (n=7) Proportion of sites with the 1 1 9 1 7 7 1 7 7 11 39 66 45 28 Abundance Alien Alien Alien Alien Alien Alien Alien Native Native Native Native Native Origin Unknown Unknown - - Generalist Generalist Generalist Generalist Generalist Generalist Generalist Generalist Generalist Generalist Generalist Generalist/A Feeding habit Dendrocerus sp Asaphes vulgaris (Walker) Aphelinus mali (Haldeman) Lysiphlebus testaceipes (Cresson) Lysiphlebus Chrysopinae sp Allograpta obliqua (Say) Allograpta exotica (Wiedemann) Pseudodoros clavatus (Fabricius) Scymnus rubicundus (Erichson) Cycloneda ancoralis (Germar) Olla v-nigrum (Mulsant) Hippodamia convergens (Guerin-Meneville) Adalia bipunctata (Mulsant) Species Harmonia axyridis (Pallas) Megaspilidae Pteromalidae Aphelinidae Braconidae Chrysopidae Syrphidae Family Coccinellidae Hymenoptera Neuroptera Diptera Order Coleoptera sites of Córdoba city, and proportion of sites where the species was recorded. sites of Córdoba city, Hyperparasitoids Parasitoids Table 1. Taxonomic classification, origin, and abundance of natural enemies associated with the aphid A. gossypii in sampling Taxonomic 1. Table Natural enemies Predators 37 Alien Species Numerically Dominate Natural Enemy Communities

Table 2. Parameters of the regression analyses performed for different variables of the natural enemy community associated with A. gossypii in relation to the urbanization index and aphid abundance. Significant and marginally significant P-values are in bold.

Urbanization index Aphid abundance Response variable

F 1,5 P R F1,5 P R

Species richness 4.32 0.09 0.46 2.03 0.21 0.29

Rarefied species richness 4.79 0.08 0.49 - - -

Species abundance 0.32 0.59 0.06 <0.01 0.99 <0.01

Shanon index 2.61 0.17 0.34 0.16 0.70 0.03

Relative richness of native species 3.30 0.13 0.40 0.98 0.37 0.16

Relative abundance of native species 1.05 0.35 0.17 <0.01 0.98 <0.01

Abundance of aphids 11.96 0.02 0.71 - - -

a 11 b 6000

10 5000

9 4000 8 3000 7 2000 Speciesrichness 6 Abundance aphids of Abundance

5 1000

4 0 -2 -1 0 1 2 3 4 5 -2 -1 0 1 2 3 4 5 Urbanization index Urbanization index

Fig. 2. Relationship between the urbanization index for Córdoba city (high values of the index mean high urbanization degree) and a) observed species richness of natural enemies of the aphid A. gossypii y = 0,56x + 7.18 and b) the abundance of A. gossypii y = 674,54x + 1817.19.

100% alien

80% native

60% Relative contribution 40%

20%

0% Richness Abundance

Fig. 3. Relative contribution (%) in terms of species richness and abundance of native and alien species of A. gossypii natural enemies considering all sampled sites in Córdoba city. 38 WAGNER, L. S., FENOGLIO, M. S., SALVO, A. DISCUSSION AND CONCLUSION Overall, our results show that urbanization does not have significant effects on the natural enemy community of the aphid A. gossypii. Two of the indicators here used to determine a-diversity of natural enemy communities (abundance and Shannon index) showed no significant changes along the urban gradient (urbanization index), which is in disagreement with our predictions. However, species richness showed a marginally significant increase with increasingly urbanized sites of the city, regardless of aphid abundance. Generalist and/or alien natural enemy species, such as those found in our study (Table 1), tend to have higher tolerance to disturbance than specialists and/or native ones (Forbes and Kendle, 1998) and therefore are likely to prevail in areas of increased urbanization (Kotze et al., 2011, Burkman and Gardiner, 2014). Moreover, studies focusing on other group of predators such as carabid communities, found an increment of species richness in urban habitats, which was attributed to the dominance of generalist and open-habitats species (Magura et al., 2004, 2010; Elek and Lovei, 2007). In our study, all the found natural enemies are generalist species (i.e. they consume more than one host/prey genus), which could explain the increase in species richness at sites of increased urbanization. Native species are usually the most negatively affected by increasing urbanization, whereas alien species may even be benefited by urban environments (McKinney, 2006). In our system, alien natural enemies were clearly dominant, mainly represented by all parasitoid and hyperparasitoid species, and most of predator species of the Coccinellinae subfamily. In a literature review, Faeth et al., (2005) found that in urban environments the relative abundance of most of the alien species show a tendency to increase at the expense of the native ones. In our work, the values of​ relative richness and abundance of native natural enemies were low (less than 41% in both cases) and similar across sites, revealing that alien species were dominant even in those city sites with low levels of urbanization. In line with these results, the similarity of the complexes of A. gossypii natural enemies between sites represented by b-diversity did not depend on the level of urbanization. In the particular case of ladybirds, the dominance of alien species was even more evident, since highly competitive alien species were present in all sites. Species like H. axyridis may be displacing (Alyokhin and Sewell, 2004; Saini, 2004; Roy et al., 2012) native species such as C. ancoralis and S. rubicundus, which were present at very low abundances in only one and two sites, respectively. Available evidence suggests that H. axyridis is an intraguild predator (Roy et al., 2012) that would cause detrimental effects on smaller species, like the native ones mentioned above (Mirande et al., 2015). Moreover, notably Eriopis connexa Germar (Coleoptera: Coccinellidae) has not been found in any of the sites, although individuals of this species were frequently collected in the city of Córdoba in previous years (as observed in labels of the several specimens deposited in the Collection of the Department of Entomology, National University of Córdoba). 39 Alien Species Numerically Dominate Natural Enemy Communities As expected, the abundance of A. gossypii increased with the level of urbanization; indeed, due to the biological characteristics of aphids (small body size, sucking mouthparts, limited mobility and multiple generations in the same host plant), and to features of the urban environment (e.g. high temperatures, stressed plants and pollution), they would benefit from disturbance (Braun and Flückiger, 1985; Raupp et al., 2010; Dale and Frank, 2014). However, the increase in prey abundance was not related to natural enemy species diversity, but we cannot completely discard an effect of urbanization mediated by the availability of resources, since the whole aphid community developing in other plant species was not sampled. In conclusion, the community of natural enemies of A. gossypii was in general not affected by urbanization, either directly or indirectly via increased food resources in jacaranda trees. The absence of a relationship between indicators of community structure and the urban gradient could be explained by the numerical dominance of alien species, which are resistant to disturbance in all urban sites. Homogenization of the biota could be occurring in the urban environment, where native species are replaced by alien ones (McKinney, 2006). However, historical data on biodiversity necessary to confirm this hypothesis (Alvey, 2006) are not available for this system. Beneficial insects play an important role as suppliers of ecosystem services such as biological pest control (Burkman and Gardiner, 2014) and have potential as biological indicators of environmental disturbance (McIntyre, 2000). Harmonia axyridis was the main natural enemy of A. gossypii in Cordoba city, but considering the invasive status of this species (Martins et al., 2009; Roy et al., 2012) it would not be suggested as a biological control agent from a diversity conservation point of view. We recommend continuing this line of research, incorporating a higher number of sampling sites in order to corroborate the present results and to provide new tools that improve both the management and conservation of these insects in urban environments.

ACKNOWLEDGEMENTS We would like to thank Leticia Zumoffen for her invaluable contribution to the identification of species of parasitoids, and María Teresa Defago for her help in field sampling. Special thanks to the anonymous reviewer for the helpful comments on the manuscript. This study was supported by MINCyT and SECyT. M.S.F. and A.S. are researchers of CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas).

REFERENCES

Alvey, A. A., 2006, Promoting and preserving biodiversity in the urban forest. Urban Forestry and Urban Greening, 5: 195-201. Alyokhin, A., Sewell, G., 2004, Changes in a lady beetle community following the establishment of three alien species. Biological Invasions, 6: 463-471. Baskin, Y., 1998, Winners and losers in a changing world. BioScience, 48: 788-792. Braun, S., Flückiger, W., 1985, Increased population of the aphid Aphis pomi at a motorway: Part 3-The effect of exhaust gases.Environmental Pollution Series A, Ecological and Biological, 39(2): 183-192. 40 WAGNER, L. S., FENOGLIO, M. S., SALVO, A.

Burkman, C. E., Gardiner, M. M., 2014, Urban greenspace composition and landscape context influence natural enemy community composition and function. Biological Control, 75: 58-67. Dale, A. G., Frank, S. D., 2014, The effects of urban warming on herbivore abundance and street tree condition. PLoS ONE, 9 (7): e102996. doi:10.1371/journal.pone.0102996 Delfino, M. A., Buffa, L. M., 2008, Afidos en plantas ornamentales de Córdoba, Argentina (Hemiptera: Aphididae). Neotropical Entomology, 37: 074-080. Delfino, M. A., Monelos, H. L., Peri P. L., Buffa, L. M., 2007, Áfidos (Hemiptera, Aphididae) de interés económico en la provincia de Santa Cruz. RIA, 36: 147-154. Denys, C., Schmidt, H., 1998, Insect communities on experimental mugwort (Artemisia vulgaris L.) plots along an urban gradient. Oecologia, 113: 269-277. Dimitri, M. J., 1980, Enciclopedia Argentina de Agricultura y Jardinería, Tercera ed. Tomo I. ACME S.A.I.C., Buenos Aires, Argentina. Dirección de Catastro Córdoba, 2007, Datos territoriales de Córdoba. http://www.cordoba.gov.ar/ cordobaciudad/principal2/default.asp?ir=55_4 25.06.2014. Elek, Z., Lövei, G. L., 2007, Patterns in ground beetle (Coleoptera: Carabidae) assemblages along an urbanisation gradient in Denmark. Acta Oecologica, 32: 104-111. Faeth, S. H., Warren, P. S., Shochat, E., Marussich, W. A., 2005, Trophic dynamics in urban communities. BioScience, 55: 399-407. Fenoglio, M. S., Salvo, A., 2010, Urbanization and parasitoids: an unexplored field of research. In: Daniels, J. A., (ed.). Advances in Environmental Research. Volume 11, Nova Science Publishers Inc., New York, 163-176. Fenoglio, M. S., Salvo, A., Estallo, E. L., 2009, Effects of urbanisation on the parasitoid community of a leafminer. Acta Oecologica, 35: 318-326. Ferrante, M., Cacciato, A. L., Lövei, G. L., 2014, Quantifying predation pressure along an urbanisation gradient in Denmark using artificial caterpillars. European Journal of Entomology, 111: 649-654. Flores Macías, A., Rodríguez Navarro, S., Ramos-Espinosa, M. G., Payán Zelaya, F., 2010, Estudio de Harmonia axirydis Pallas (Coleoptera: Cocinellidae) como bioagente de control de Aphis gossypii Glover (Hemiptera: Aphididae). Interciencia, 35: 506-509. Forbes, S., Kendle, T., 1998, Urban Nature Conservation: Landscape Management in the Urban Countryside. Taylor & Francis, London, UK, 368. Frankie, G. W., Ehler, L. E., 1978, Ecology of insects in urban environments. Annual Review of Entomology, 23: 367-387. Gonzalez, G., 2006, Los Coccinellidae de Chile (online). http://www.coccinellidae.cl (01.09.2014). Graham, M. H., 2003, Confronting multicollinearity in ecological multiple regression. Ecology, 84: 2809-2815. Hunter, M. D., 2002, Landscape structure, habitat fragmentation, and the ecology of insects. Agricultural and Forest Entomology, 4: 159-166. INDEC, 2010, Censo Nacional de Población, Hogares y Viviendas 2010: total del país, resultados previsionales. 1 ed, Buenos Aires. http://censo2010.indec.gov.ar/preliminares/cuadro_cordoba.asp Jones, E. L., Leather S. R., 2012, Invertebrates in urban areas: a review. European Journal of Entomology, 109: 463-478. Koricheva, J., 1994, Can parasitoids explain density patterns of Eriocrania (Lepidoptera: Eriocraniidae) miners in a polluted area? Acta Oecologica, 15: 365-378. Kotze, J., Venn, S., Niemelä, J., Spence, J., 2011, Effects of urbanization on the ecology and evolution of arthropods. In: Niemelä, J., (Ed.). Urban Ecology, Patterns, Processes and Applications. Oxford University Press, New York, USA, 159-166. Legendre, P., Legendre, L., 1998, Numerical Ecology, 2nd English edn. Elsevier Science BV, Amsterdam, 853. 41 Alien Species Numerically Dominate Natural Enemy Communities

López, R., Araya, J. E., Sazo, L., 2012, Colectas de Syrphidae (Diptera) en alfalfa en Colina, Región Metropolitana, Chile, y clave de identificación de seis especies de Allograpta. Boletín de Sanidad Vegetal Plagas, 38: 3-15. Losey, J. E., Vaughan, M., 2006, The economic value of ecological services provided by insects. Bioscience, 56: 311-323. Magura, T., Lövei, G. L., Tóthmérész, B., 2010, Does urbanization decrease diversity in ground beetle (Carabidae) assemblages? Global Ecology and Biogeography, 19: 16-26. Magura, T., Tóthmérész, B., Molnár, T., 2004, Changes in carabid beetle assemblages along an urbanisation gradient in the city of Debrecen, Hungary. Landscape Ecology, 19: 747-759. Magurran, A. E., 2004, Measuring Bbiological Diversity. Blackwell Publishing, Malden, USA, 215. Margaritopoulos, J. T., Tzortzi, M., Zarpas, K. D., Tsitsipis, J. A., Blackman, R. L., 2006, Morphological discrimination of Aphis gossypii (Hemiptera: Aphididae) populations feeding on Compositae. Bulletin of Entomological Research, 96: 153-165. Martins, C. B., Almeida, L. M., Zonta-De-Carvalho, R. C., Castro, C. F., Pereira, R. A., 2009, Harmonia axyridis: a threat to Brazilian Coccinellidae? Revista Brasileira de Entomologia, 53: 663-671. Martinson, H. M., Raupp, M. J., 2013, A meta-analysis of the effects of urbanization on ground beetle communities. Ecosphere 4: 60 http://dx.doi.org/10.1890/ES12-00262.1 McDonald, R. I., Marcotullio, P. J., Güneralp, B., 2013, Urbanization and global trends in biodiversity and ecosystem services. In: Elmqvist, T., et al., (Eds.). Urbanization, Biodiversity and Ecosystem Services: Challenges and Opportunities. A global assessment Springer, The Netherlands, 31-53. McIntyre, N. E., 2000, Ecology of urban arthropods: a review and a call to action. Annals of the Entomological Society of America, 93: 825-835. McKinney, M. L., 2002, Urbanization, Biodiversity, and Conservation. The impacts of urbanization on native species are poorly studied, but educating a highly urbanized human population about these impacts can greatly improve species conservation in all ecosystems. BioScience, 52: 883-890. McKinney, M. L., 2006, Urbanization as a major cause of biotic homogenization. Biological Conservation, 127: 247-260. McKinney, M. L., 2008, Effects of urbanization on species richness: a review of plants and animals.Urban Ecosystems, 11: 161-176. McKinney, M. L., Lockwood, J. L., 1999, Biotic homogenization: a few winners replacing many losers in the next mass extinction. Trends in Ecology and Evolution, 14: 450-453. Mirande, L., Desneux, N., Haramboure, M., Schneider, M. I., 2015, Intraguild predation between an alien and a native coccinellid in Argentina: the role of prey density. Journal of Pest Science, 88: 155-162. Niemelä, J., 1999, Is there a need for a theory of urban ecology? Urban Ecosystems, 3: 57-65. Pickett, S. T. A., Cadenasso, M. L., Grove, J. M., Boone, C. G., Groffman, P. M., Irwin, E., Kaushal, S. S., Marshall, V., Mcgrath, B. P., Nilon, C. H., Pouyat, R. V., Szlavecz, K., Troy, A., Warren, P., 2011, Urban ecological systems: Scientific foundations and a decade of progress.Journal of Environmental Management, 92: 331-362. R Core Team, 2012, R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org/ Raupp, M. J., Shrewsbury, P. M., Herms, D. A., 2010, Ecology of herbivorous arthropods in urban landscapes. Annual Review of Entomology, 55: 19-38. Roy, H. E., Adriaens, T., Isaac, N. J. B., Kenis, M., Onkelinx, T., San Martin, G., Brown P. M. J., Hautier, L., Poland, R., Roy, D. B., Comont, R., Eschen, R., Frost, R., Zindel, R., Van Vlaenderen, J., Nedved, O., Ravn, H. P., Grégoire, J. C., Biseau, J. C., Maes, D., 2012, Invasive alien predator causes rapid declines of native European ladybirds. Diversity and Distributions, 18: 717-725. Saini, E. D., 2004, Presencia de Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) en la provincia de Buenos Aires. Aspectos biológicos y morfológicos. RIA, 33: 151-160. 42 WAGNER, L. S., FENOGLIO, M. S., SALVO, A.

Weems, H. V., 2004, A Hover Fly, Allograpta obliqua (Say) (Insecta: Diptera: Syrphidae). University of Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, EDIS. Yokomi, R. K., Tang, Y. Q., 1996, A survey of parasitoids of brown citrus aphid (Homoptera: Aphididae) in Puerto Rico. Biological Control, 6: 222-225. Zanette, L. R. S., Martins, R. P., Ribeiro, S. P., 2005, Effects of urbanization on Neotropical wasp and bee assemblages in a Brazilian metropolis. Landscape and Urban Planning, 71: 105-121. Zumoffen, L., Rodriguez, M., Gerding, M., Salto, C. E., Salvo, A., 2015, Plantas, áfidos y parasitoides: interacciones tróficas en agroecosistemas de la provincia de Santa Fe, Argentina y clave para la identificación de los Aphidiinae y Aphelinidae (Hymenoptera) conocidos para la región. Revista de la Sociedad Entomológica Argentina, 74: 133-144.

Received: December 09, 2015 Accepted: June 21, 2017 J. Entomol. Res. Soc., 19(2): 43-52, 2017 ISSN:1302-0250

Do Beetles Prefer the Odor of Female-Stage to Male-Stage Flowers in Atemoya, a Cantharophylous Protogynous Fruit Tree (Annonaceae)?

Morio TSUKADA1* Miki INUI1 Noritaka SUZAKI2

1 Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, JAPAN 2 Kinan Fruit Tree Science Branch, Mie Prefecture Agricultural Research Institute, Mihama, Mie 519-5202, JAPAN, e-mails: *[email protected] , [email protected]

ABSTRACT The flowers of annonaceous fruit trees cherimoya (Annona cherimola) and atemoya (A. cherimola x squamosa) exhibit protogynous dichogamy. Their main pollinators are sap beetles (Coleoptera: Nitidulidae). However, pollination by beetles is usually not sufficient for commercial fruit production, so costly hand pollination is required in many areas. These beetles on cherimoya and atemoya are thought to visit the female-stage flower, remain inside it, and leave the flower when it has transitioned to the male stage; however, no study has yet directly elucidated the visiting behavior of the beetles. In this study, we examined this hypothesis using olfactometer testing in the field. Both male and female sap beetles, Carpophilus marginellus, were significantly attracted to the odor of female-stage flowers, but not to the odor of late male-stage flowers. We conclude that the beetles prefer the odor of female-stage flowers to that of late male-stage flowers. These findings support the above hypothesis describing beetle pollination behavior. Key words: Annonaceae, Nitidulidae, floral odor, beetle pollination, behavior.

INTRODUCTION The custard apple, or cherimoya, Annona cherimola Miller, the sugar apple, A. squamosa Linnaeus, and their hybrid, atemoya (Annona cherimola x squamosa) are cultivated for their fruits, especially in warm regions, worldwide. Cherimoya originates from the highlands of Peru, whereas the sugar apple originates from Caribbean islands. They belong to the family Annonaceae, a mainly tropical plant taxa of the order Magnoliales, which is relatively primitive in angiosperm (Moore et al., 2007). A majority of studied plants of the family exhibit protogynous dichogamy and employ beetles as pollination agents (Gottsberger, 1974; 1988; 1989a; 1989b; Armstrong and Irvine, 1989; Andrade et al., 1996; Dieringer et al., 1999; Bernhardt, 2000; Endress, 2010). Annona fruit trees are also protogynous and known to be cantharophilous (González and Cuevas, 2011). The timing of the change of sex from female to male varies with locality and variety (Nadel and Peña, 1994; González and Cuevas, 2011; Kishore et al., 2012). In Israel, atemoya flowers enter the female stage in the evening, and transition to the male stage approximately 24h later (Podoler et al., 1984). On the 44 TSUKADA, M., INUI, M., SUZAKI, N. other hand, in Florida, atemoya flowers open in mid- to late afternoon, and become male around noon the next day (Nadel and Peña, 1994). González and Cuevas (2011) report that in Spain, cherimoya anthesis occurs gradually from early afternoon, the flowers transition to the male stage 24h later. In Japan, the sexual phase usually changes from 16:00 to 21:00 in both cherimoya and atemoya, although a small proportion of atemoya flowers become male in the morning. Because the female and male stages do not normally overlap, natural fruit set without pollinators is generally low (Richardson and Anderson, 1996). As is usual in the family Annonaceae (Okada, 1993), Annona flowers have no nectary. In addition, they have green petals, and only small openings between the petals during the female stage (Podoler et al., 1985; González and Cuevas, 2011). As a result, attempts to use honeybees or bumblebees for pollination have failed, leading to the need for costly hand pollination in places where natural fruit set is not sufficient (Saavedra, 1977; George and Campbell, 1991; Richardson and Anderson, 1996). The long search for effective pollination agents continues in agricultural scene. Surveys on natural pollinators for Annona fruit trees have shown that the flowers rely mainly on beetles of the family Nitidulidae (Gazit et al., 1982; George et al., 1989; Nadel and Peña, 1994; Nagel et al., 1989; Blanche and Cunningham, 2005; Jenkins et al., 2013; but see also Caleca et al., 1996; 1998) for pollination. A periodical collection of flower visitors in sugarapple and atemoya orchard in Florida suggested that insects are attracted to female-stage flowers, and remain there until the flowers become male (Nadel and Peña, 1994). The insects are assumed to be released from the flower with pollen grains adhering to their body surface (Fig. 1; Gazit et al., 1982). At this time, new flowers enter the female stage to which the insects with pollen are attracted, and pollen grains will in this way be deposited on their stigmata. This hypothesis is accepted in many studies. However, the pioneer work of Podoler et al. (1984) showed that experimentally cut atemoya flowers attracted beetles in the morning and evening at the female and the early male stage, respectively, and that they lose their attractiveness in the late male stage. This does not necessarily support the hypothesis, because when the late male-stage flowers have lost their odor, female-stage flowers may also have lost their odor. Thus, the movement of beetles from male- to female-stage flowers may be prevented. However, until now, no study has observed the actual behavior of the beetles; i.e., whether they prefer female-stage flowers to late male-stage ones in these fruit trees. Although there is no evidence that flowers offer mating site for beetles in chrimoya, atemoya and sugarapple (Peña et al., 2002), many beetle-pollinated flowers offer mating site for the visitors (e.g., Thien et al., 1985; Tang, 1987; Johnson and Midgley, 2001). In addition, males of many nitidulid beetles especialy in the genus Carpophilus produce aggregation pheromones which attract both sexes. Therefore, even if only the male beetles was attracted to the flower odor, both sexes can be in the flower because the male individuals attract another individuals of both sexes by the aggregation pheromone. However, this scenario has not been tested in previous field studies. 45 Do Beetles Prefer the Odor of Female-Stage to Male-Stage Tsukada et al., (2005) reported 29 species of insects from cherimoya flowers in orchards in Wakayama Prefecture, Japan. Of these, 7 species were nitidulid beetles. Higuchi et al. (2014b) studied the pollination ability of orchard-caught beetles, and concluded that Haptoncus ocularis (Fairmaire) (Nitidulidae) and Mimemodes monstrosus (Reitter) (Rhizophagidae) can pollinate cherimoya. Further, Higuchi et al. (2014a) revealed mass-reared H. ocuralis, M. monstrosus, and Carpophilus marginellus Motschulsky (Nitidulidae) to have the ability to pollinate cherimoya, even if reared exclusively on pineapple for several generations. These insects therefore retain the ability to respond to the odor of cherimoya flowers. In the present study, we investigated whether the female- and late male-stage flower at the same time of a day attract C. marginellus adults so that the pollen can be transferred from male-stage flowers to female-stage ones on atemoya trees. Furthermore, we sexed the tested beetles to test if only male or both sexes respond to the odor.

Fig. 1. Flowering and the hypothesized pollination pattern by beetles in Annona spp.

MATERIALS AND METHODS

Study sites and materials Atemoya trees were cultivated at the Kinan Fruit Tree Science Branch of Mie Prefecture Agricultural Research Institute, Mie, Japan, and in a commercial orchard on Ishigaki Island, Okinawa, Japan. These trees were used for in situ preference experiments in the summer of 2007, from June to August in Mie, and in November in Okinawa. The Mie site comprised five atemoya trees planted in a greenhouse. The Okinawa site was an open orchard, with 13 atemoya trees planted alongside other fruit trees. The atemoya flowers in our study sites emitted a fruit-like odor in the evenings, during both the female and male stages. In some cases, the flowers emitted odor even in the morning. Laboratory stock of the sap beetle C. marginellus was used for the preference test. This stock was founded from individuals caught in atemoya flowers on the campus of Mie University, Tsu, Mie, Japan. They were kept at 25 ˚C, 16L:8D conditions with 46 TSUKADA, M., INUI, M., SUZAKI, N. cut pineapple as food. See Tsukada et al. (2008) for details of rearing and larval development. The insects were prepared as follows. 1) Last-instar larvae were selected from the stock. 2) These larvae were individually kept in small vials until eclosion, with wet tissue paper providing the pupation site. 3) Newly-emerged adults were supplied with water for 3 - 5 days, the required period for sexual maturation, until the experiment.

Experiments T-shaped glass tubes, 8 mm inner diameter, 70 mm in length (the vertical part of the T) and 110 mm wide (the horizontal part of the T) were connected with transparent vinyl chloride tube (12 mm inner diameter) at the three ends (Fig. 2). The vinyl tube from the lower end of the glass tube was connected to an air pump (Shibata Mini Pump MP-2N), which vented the air from the tube. This vinyl tube was readily disconnected from the T-tube to allow placement of a test insect in the tube.

Fig. 2. Outline of the T-shape olfactometer. Insects were individually placed in the T-shape glass tube and allowed to choose one of the two ends. Contrary to the results of Podoler et al. (1984), who carried out preference experiments using cut atemoya flowers, our preliminary experiments showed that cut flowers have a repellant effect on the beetles. We therefore did not cut them: instead, a flower on a tree was gently covered with a plastic bag measuring ca. 30 x 20 cm. Openings on the bag allowed ventilation. One of the vinyl tubes from an upper end of the glass tube was connected to the flower on the tree. The last tube was led to outside of the greenhouse as a control or was connected to another flower. The experiments were always carried out from 19:00 - 22:00, after sunset. The ambient temperature was 20 - 26 ˚C. We expected the pollinator beetles to move from late male-stage flowers to a female-stage flowers in this period because during this time, the male-stage flower gradually loses its odor, whereas the female-stage flower may still emit a fruit-like odor. Previous studies have suggested that both female and male-stage flowers emit a fruit-like odor (Podoleret al., 1984), and beetles which entered during the female stage leave the flower late after the flower enters the male stage (Nadel and Peña, 1994). Our preliminary observations supported these observations. 47 Do Beetles Prefer the Odor of Female-Stage to Male-Stage Combinations of female-stage flower vs. control, male-stage flower vs. control, and female-stage flower vs. male-stage flower were tested for beetle attraction. The flow of air was regulated, using flow meters, at 0.5 - 0.7 l / min at the lower end of the T-tube. Insects were gently placed in the T-tube from the bottom individually, and observed for 5 min. When the insect reached one of the upper ends of the T-tube, their choice was recorded and the trial ended. All the flowers and insects were used for the experiment only once. The number of trials with each combination was about 20. The tubes were replaced every time and carefully washed with water and ethanol, then dried naturally before the next use. Voucher specimens are deposited in the Insect Ecology Laboratory of Mie University. The obtained data were analyzed using a binomial test for the difference within the sex of beetle, and by Fisher’s Exact test for the difference in behavior between the two sexes of the beetle. The tests were performed with R 2.9.2 software (R Development Core Team, 2010).

RESULTS In five of the total of 117 trials, the insects did not choose either of the two ends of the T-tube within 5 min. In consideration of this relatively small number, we omitted them from the analyses. Both male and female individuals were significantly more likely to choose female-stage flower to controls (Fig. 3;p < 0.01, p < 0.001, respectively). Although male insects made more definitive choices than did the females, there was no significant difference between male and female insects. For male-stage flowers, both male and female insects showed no statistically significant choices (Fig. 4). Although male insects were slightly more likely to choose flowers, while female insects were more likely to choose the control, there was no statistically significant difference in this behavior between the two insect sexes. Finally, for the test between the flowers in the two stages, female-stage flowers were chosen over male-stage flowers by both male and female insects (Fig. 5; p < 0.001, p < 0.05, respectively). There was no difference in this behavior between the two sexes of the insect.

100 N=19 N=15 80 Female flower

Control 60

40

% choice by by % choice beetles 20

0 Male beetle Female beetle

Fig. 3. Results of the choice test by male and female C. marginellus to the odor of a female flower or control. 48 TSUKADA, M., INUI, M., SUZAKI, N.

100

80 N=18 N=19 Male 60 flower Control

40

% choice by by % choice beetles 20

0 Male beetle Female beetle

Fig. 4. Results of the choice test by male and female C. marginellus to the odor of a male flower or control.

100 N=17

80 N=23 Female flower 60 Male flower

40

20 % choice by by % choice beetles

0 Male beetle Female beetle

Fig. 5. Results of the choice test by male and female C. marginellus to the odor of a female flower and a male flower.

DISCUSSION Our results clearly show that C. marginellus adults are attracted to the odor of female-stage flowers of atemoya in the evening, but at that time, they are not attracted to the odor of late-male-stage flowers. These results are consistent with the previous hypothesis, that in the atemoya and its relative’s pollination system, beetles are attracted to the flower during the female stage, remain in the flower, and are released during the male stage with pollen grains adhering to the surface of their body. The released beetles are then assumed to visit a new female-stage flower (Nadel and Peña, 1994). However, early male-stage flowers emit a strong odor that can also attract beetles (Podoler et al., 1984). Furthermore, Nadel and Peña (1994) noted that beetles do not escape from the male flowers immediately after the sexual phase change. Our preliminary observations showed the same results (unpublished). Nevertheless, this study has shown the conventional hypothesis of pollination for Annona fruit trees to be most likely true. Even though a floral odor is emitted from female and male-stage flowers from evening to early night, the timing of the disappearance of the odor might be slightly different; i.e., emission of odor during the female stage lasts longer, whereas that during the male stage ceases sooner. This difference appears to motivate beetles to move from male-stage flowers to female-stage flowers. Therefore, breeding and/or 49 Do Beetles Prefer the Odor of Female-Stage to Male-Stage cultivation techniques that ensure the disappearance of male odor while the female flower emits a strong odor may improve fruit set by beetle pollination. Some cycad and relatively primitive angiosperms employ a push-pull or similar strategy to control insect behavior and achieve effective pollination (Terryet al., 2007; Teichert et al., 2011). Protogynous flowers typically emit an attractive odor during the female stage and emit a repelling odor at the late female stage, and then emit an attractive odor again in the male stage (Terry et al., 2007). In this study, however, we found no repellant effect of late male-stage floral odor. Instead, the attractiveness of the late male-floral odor was the same as that of the control (ambient air) (Fig. 2). We therefore suggest that the strong odor in the early male stage simply fades with the elapse of time. The results of this study support the conventional hypothesis on beetle pollination behavior for cherimoya and atemoya (Gazit et al., 1982; Nadel and Peña, 1994). However, atemoya flowers seem to emit their odor usually only in the evening to the early night (Podoler et al., 1984), and occasionally in the morning (Tsukada, unpublished data). The flower apparently provides no rewards such as nectar or warmth. On the other hand, Podoler et al. (1985) recorded the longevity of a pollinator, Carpophilus hemipterus, to be greater with experience of contact with flowers than without. The reason for this has not been clarified. However, if this is applicable for all visitors, it might be the reason why the beetles stay for long periods in the flower after they have been attracted to the female-stage flowers. Beetle attraction by floral odor is found also in other genera of the family Annonaceae (Andrade et al., 1996; Bernhardt, 2000; Gottsberger, 1977; 1989a; 1989b; 1999; 2012; Jürgens et al., 2000; Maia et al., 2012). In Asimina, the flower emits an odor that consists chiefly of esters, typical of fruit odors (Goodrich and Raguso, 2009). Since most flower-visiting nitidulid beetles found inAnnona flowers are frugivorous (George et al., 1992; Tsukada et al., 2005), it is likely that flowers chemically mimic the odor of rotting fruit to lure frugivorous beetles (Peña et al., 2002; Goodrich et al., 2006). We found no significant difference between the two sexes of beetles in terms of attraction to the floral odor. Therefore, it is unlikely that males visit the flowers first by responding to the odor, and attract other individuals by their aggregation pheromone. Rather, beetles are attracted to the floral odor regardless of their sex. This can be confirmed by examining the distribution of beetles of each sex in flowers, in the field. In the family Annonaceae, which are relatively primitive angiosperms, beetle pollination is most common, although thrips, flies, cockroaches and other insect species also pollinate certain plant species (Jürgens et al., 2000; Nagamitsu and Inoue, 1997; Saunders, 2012). Molecular phylogeny shows that pollination by small beetles such as nitidulid beetles is an ancestral trait in Annonaceae (Saunders, 2012): in the gymnosperm family Cycadaceae, which is a neighbor species to angiosperms (Schneider et al., 2002), beetles appear to be the most frequent pollinators (Kono and Tobe, 2007; Procheş and Johnson, 2009). Therefore, the sophisticated pollination system seen in Annona spp., although not specialized, seems to be common in 50 TSUKADA, M., INUI, M., SUZAKI, N. the early era of angiosperm evolution. However, because atemoya is a hybrid of cherimoya and sugar apple (Morton, 1987) that was developed only a century ago, natural selection is unlikely to have shaped the flowering behavior of this plant. Careful observation of sugar apple and cherimoya should therefore provide more information and lead to a better understanding of the evolution of pollination in Annona spp.

ACKNOWLEDGEMENTS We thank Dr. H. Higuchi of Kyoto University and Dr. Y. Yonemoto of Japan International Research Center for Agricultural Sciences for their kind advises on biology of Annona plants during the study. Mr. H. Inoue of Okinawa Prefectural Agricultural Experimental Station always supported our study in Ishigaki. Mr. and Ms. Kinjo kindly allowed us to use their orchard for this experiment. Comments on earlier draft by Prof. Gottsberger and an anonymous referee improved the manuscript. This work was supported by JSPS KAKENHI Grant Numbers 18580052 and 21580065 to MT.

REFERENCES

Andrade, B. M., Oliveira-Filho, A. T., Soares, A. R., 1996, Pollination and breeding system of Xylopoa brasiliensis Sprengel (Annonaceae) in south-wastern Brazil. Journal of Tropical Biology, 12: 313-320. Armstrong, J. H., Irvine, A. K., 1989, Floral biology of Myristica insipida (Myristicaceae), a distinctive beetle pollination syndrome. American Journal of Botany, 76: 86-94. Bernhardt, P., 2000, Convergent evolution and adaptive radiation of beetle-pollinated angiosperms. Plant Systematics and Evolution, 222: 293-320. Blanche, R., Cunningham, S. A., 2005, Rain forest provides pollinating beetles for atemoya crops. Journal of Economic Entomology 98: 1193-1201. Caleca, V., Verde, G. L., Chihara, S. R. D., Sinacori, A., Tsolakis, H., 1996, Observations on insects visiting flowers of cherimoya and atemoya in Sicily (Italy).Bollettino di Zoologia Agraria e di Bachicoltura, 28: 185-194. Caleca, V., Verde, G. L., Chihara, S. R. D., Tsolakis, H., 1998, Effects of releases of Orius laevigatus (Fieber) (Rhynchota Anthocoridae) and hand pollination on Cherimoya (Annona cherimola Mill.) fruit production in Sicily. Bollettino di Zoologia Agraria e di Bachicoltura, 30: 207-212. Dieringer, G., Cabrera, R. L., Lara, M., Loya, L., Reyes-Castillo, P., 1999, Beetle pollination and floral thermogenicity in Magnolia tamaulipana (Magnoliaceae). International Journal of Plant Science, 160: 64-71. Endress, P., 2010, The evolution of floral biology in basal angiosperms. Philosophical Transactionf of the Royal Society B, 365: 411-421. Gazit, S., Galon, I., Podoler, H., 1982, The role of Nitidulid beetles in natural pollination of Annona in Israel. Journal of the American Society for Horticultural Science, 107: 849-852. George, A. P., Campbell, J. A., 1991, Hand pollination in subtropical Australia. California Rare Fruit Growers, 15: 36-38. George, A. P., Nissen, R. J., Campbell, J. A., 1992, Pollination and selection in Annona species (cherimoya, atemoya and sugar apple). Acta Horticulturae, 321: 178-185. George, A. P., Nissen, R. J., Ironside, D. A., Anderson, P., 1989, Effects of nitidulid beetles on pollination and fruit set of Annona spp. hybrids. Scientia Horticulturae Amsterdam, 39: 289-299. González, M., Cuevas, J., 2011, Reproductive barriers in Annona cherimola (Mill.) outside of its native area. Plant Systematics and Evolution 297: 227-235. 51 Do Beetles Prefer the Odor of Female-Stage to Male-Stage

Goodrich, K. R., Raguso, R. A., 2009, The olfactory component of floral display in Asimina and Deeringothamnus (Annonaceae). New Phytologist, 183: 457-469. Goodrich, K. R., Zjhra, M. L., Ley, C. A., Raguso, R. A., 2006, When flowers smell fermented: the chemistry and ontogeny of yeasty floral scent in pawpaw Asimina triloba: annonaceae. International Journal of Plant Science, 167: 33-46. Gottsberger, G., 1974, The structure and function of the primitive angiosperm flower - a discussion. Acta Botanica Neerlandica, 23: 461-471. Gottsberger, G., 1977, Some aspects of beetle pollination in the evolution of flowring plants. Plant Systematics and Evolution, Supplement, 1: 211-226. Gottsberger, G., 1988, The reproductive biology of primitive angiosperms. Taxon, 37: 630-643. Gottsberger, G., 1989a, Beetle pollination and flowering rhythm of Annona spp. (Annonaceae) in Brazil. Plant Systematics and Evolution, 167: 165-187. Gottsberger, G., 1989b, Comments on flower evolution and beetle pollination in the genera Annona and Rollinia (Annonaceae). Plant Systematics and Evolution, 167: 189-194. Gottsberger, G., 1999, Pollination and evolution in neotropical Annonaceae. Plant Species Biology 14: 143-152 Gottsberger, G., 2012, How diverse are Annonaceae with regard to pollination? Botanical Journal of the Linnean Society, 169: 245-261. Higuchi, H., Tsukada, M., Ninomiya, T., Furukawa, T., Yoshida, A., Kuriyama, M., 2014a, Pollination of cherimoya (Annona cherimola Mill.) by the mass-propagated beetles Haptoncus ocularis, Mimemodes monstrosus, and Carpophilus marginellus. Tropical Agriculture and Development, 58: 18-25. Higuchi, H., Tsukada, M., Yoshida, A., Furukawa, T., 2014b, Effective pollinators among Japanese fauna of the insect visitors of cherimoya (Annona cherimola Mill.). Tropical Agriculture and Development 58: 33-36. Jenkins, D. A., Cline, A. R., Irish B., Goenaga, R., 2013, Attraction of pollinators to atemoya (Magnoliales: Annonaceae) in Puerto Rico: A synergistic approach using multiple nitidulid lures. Journal of Economic Entomology, 106: 305-310. Johnson, S. D., Midgley, J. J., 2001, Pollination by monkey beetles (Scarabaeidae: Hopliini): Do color and dark centers of flowers influence alighting behavior? Environmental Entomology, 30: 861-868. Jürgens, A., Webber, A. C., Gottsberger, G., 2000, Floral scent components of Amazonian Annonaceae species pollinated by small beetles and thrips. Phytochemistry, 55: 551-558. Kishore, K., Shukla, A. K., Babu, N., Sarangi, D. N., Patanayak, S., 2012, Pollination biology of Annona squamosa L. (Annonaceae): Evidence for pollination syndrome. Scientia Horticulturae Amsterdam, 144: 212-217. Kono, M., Tobe, H., 2007, Is Cycas revoluta (Cycadaceae) wind- or insect-pollinated? American Journal of Botany, 94(5): 847-855. Maia, A. C. D., Dotterl, S., Kaiser, R., S-Gottsberger, I., Teichert, H., Gibernau, M., Navarro, D. M. A. F., Schlindwein, C., Gottsberger, G., 2012, The key role of 4-methyl-5-vinylthiazole in the attraction of scarab beetle pollinators: a unique olfactory floral signal shared by Annonaceae and Araceae.Journal of Chemical Ecology, 38 1072-1080. Moore, M. J. Bell, C. D., Soltis, P. S. Soltis, D. E., 2007, Using plastid genome-scale data to resolve enigmatic relationships among basal angiosperms. Proceedings of the National Academy of the USA 104(49): 19363-19368. Morton, J. F., 1987, Fruits of Warm Climates. Florida Flair Books, Miami, Florida, 505. Nadel, H., Peña, J. E., 1994, Identity, behavior, and efficacy of nitidulid beetles (Coleoptera: Nitidulidae) pollinating commercial Annona species in Florida. Environmental Entomology, 23: 878-886. Nagamitsu, T., Inoue, T., 1997, Cockroach pollination and breeding system of Uvaria elmeri (Annonaceae) in a lowland mixed-dipterocarp forest in Sarawak. American Journal of Botany, 84: 208-213. 52 TSUKADA, M., INUI, M., SUZAKI, N.

Nagel J., Peña, J. E., Habeck, D., 1989, Insect pollination of atemoya in Florida. Florida Entomologist, 72: 207-211. Okada, H., 1993, Reproductive biology of annonaceous plants. Plant Species Biology, 17: 1-11 (in Japanese). Peña, J. E., Nadel, H., Barbosa-Pereira, M., Smith, D., 2002, Pollinators and pest of Annona species. In: Peña, J. E., Sharp, J. L., Wysoki, M. (Eds.), Tropical fruit pests and pollinators: biology, economic importance, natural enemies and control. Wallingford: CABI International, 197-221. Podoler, H., Galon, I., Gazit, S., 1984, The role of nitidulid beetles in natural pollination of annona in Israel. Acta Oecologica, Oeccologia Applicata, 5(4): 369-381. Podoler, H., Galon, I., Gazit, S., 1985, The effect of atemoya flowers on their pollinators: nitidulid beetles. Acta Oecologica, Oecologia Applicata, 6(3): 251-258. Procheş, S., Johnson, S. D., 2009, Beetle pollination of the fruit-scented cones of the South African cycad Staingeria eriopus. American Journal of Botany, 96(9): 1722-1730. R Development Core Team, 2010, R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org. Richardson, A. C., Anderson, P. A., 1996, Hand pollination effects on the set and development of cherimoya (Annona cherimola) fruit in a humid climate. Scientia Horticulturae Amsterdam, 65(4): 273-281. Saavedra, 1977, Influence of pollen grain stage at the time of hand pollination as a factor on fruit set of cherimoya. Hortscience, 12(2): 117-118. Saunders, R. M. K., 2012, The diversity and evolution of pollination systems in Annonaceae. Botanical Journal of the Linnean Society, 169(1): 222-244. Schneider, D., Wink, M., Sporer, F., Lounibos, P., 2002, Cycads: their evolution, toxins, herbivores and insect pollinators. Naturwissenschaften, 89(7): 281-284. Tang, W., 1987, Insect pollination in the cycad Zamia pumila (Zamiaceae). American Journal of Botany 74(1): 90-99. Teichert, H., Dötterl, S., Gottsberger, G., 2011, Heterodichogamy and nitidulid beetle pollination in Anaxagorea prinoides, an early divergent Annonaceae. Plant Systematics and Evolution, 291(1): 25-33. Terry, I., Walter, G. H., Moore, C., Roenmer, R., Hull, C., 2007, Odor-mediated push-pull pollination in cycads. Science, 318: 70. Thien, L. B., Bernhardt, P., Gibbs, G. W., Pellmyer, O., Bergstron, G., Groth I., McPherson, G., 1985, The pollination of Zygogynum (Winteraceae) by a moth, Sabatinca (Micropterigidae): and ancient association? Science, 227: 540-543. Tsukada, M., Higuchi, H., Furukawa, T., Yoshida, A., 2005, Flower Visitors to Cherimoya, Annona cherimola (Magnoliales: Annonaceae) in Japan. Applied Entomology and Zoology, 40(2): 317-324. Tsukada, M., Tanaka, D., Higuchi, H., 2008, Thermal requirement for development of Carpophilus marginellus (Coleoptera: Nitidulidae), a potential pollinator of cherimoya and atemoya trees (Magnoliales: Annonaceae). Applied Entomology and Zoology, 43(2): 281-285.

Received: January 07, 2016 Accepted: July 27, 2016 J. Entomol. Res. Soc., 19(2): 53-65, 2017 ISSN:1302-0250

Impact of Gamma Radiation on Male Proboscis of Rhynchophorous ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae)

Eman Ahmed MAHMOUD1 Ahlam GABARTY2*

1Biological Applications Dept., Nuclear Research Center, Atomic Energy Authority, Cairo, EGYPT 2*Natural Products Research Dept., National Center for Radiation Research and Technology, Atomic Energy Authority, Cairo, EGYPT, e-mail: [email protected]

ABSTRACT Red palm weevil, Rhynchophorous ferrugineus (Olivier, 1790), (Coleoptera: Curculionidae) is the most dangerous pest for date palms in the Middle East, especially the Arab Gulf countries. The proboscis sensilla in males are studied by using Scanning Electron Microscopy (SEM) to assess the effect of gamma irradiation with different doses. Eight types of sensilla have been observed; four types of coeloconic sensilla (I, II, III, IV) are distributed on the lateral and frontal cuticular surface of the proboscis with a chemoreceptors function. Trichodea sensilla (I, II) and coeloconic sensilla (V) are concentrated on the dorsal surface of proboscis and are arranged in two rows to serve an olfactory and mechanosensory function. A very few number of squamiform sensilla (I) are scattered on the anterior-lateral side of proboscis to perform a mechanoreception function. Such malformation that gave rise to morphological structures of sensilla after exposing the pupae to the doses 10, 15, and 20 gray of gamma radiation are discussed. Irradiation with dose 10 Gy had a slight effect on the different types of sensilla as compared to the control, whereas at the radiation doses (15, 20 Gy) percentage of malformation are increased. The information gained from this study shows that the doses 15, 20 Gy have affected most types of sensilla and that the coeloconic sensilla (IV) are the only type that is not affected by any dose of irradiation. Key words: Gamma radiation, R. ferrugineus, proboscis, sensilla.

INTRODUCTION Infestations of red palm weevil (RPW) have been reported in over 50% of the date palm growing countries, sparing none in the Middle East (Faleiro, 2006). Injured trees release highly volatile compounds that attract male red palm weevils (Gunawardena et al., 1998). After their arrival, males secrete aggregation pheromones that attract all males and females. Transfer of date palm offshoots as planting material has played a major role in rapid proliferation of the pest in the Middle East (Abraham et al., 1998). RPW is considered as a wound parasite because infestation usually starts in the wounds. One of the main causes of wounds is the detachment of offshoots from the mother tree. Infestations, therefore, mostly occur in the lower part of the trunk, less than one-meter above the soil surface (Lukmah and Alquat, 2002). 54 MAHMOUD, E. A., GABARTY, A. Chemical communication between insects and host plants is one of the aspects of insect chemoecology. By clarifying the mechanism of chemical communication between an insect and its host plant, chemoecological research provides the theoretical basis for integrated management. Receptors are the main tools of insect chemical communications and are mainly located in the antenna (Liu, 2006). Adult antennae of many insect species possess various types of sensilla with different functions that play important roles in various behaviors during adult life (Gao et al., 2007). Antennal sensilla, detecting various stimuli, are implicated in the recognition of food, hosts, or partners (Onagbola et al., 2008). Receptors adjust feeding preferences (De Bore, 2006), recognize host plant odors (Skiri et al., 2005), and play important roles in insect survival. The types of sensilla on head and rostrum of RPW were studied by Sharaby and Al-Dosary (2006). They identified three types of sensilla; trichoid sensilla I, II and coeloconic sensilla. These sensilla may be recognized as pheromone-receptors or mechanoreceptors. The main purpose of this paper is to describe the divergence in morphological characteristics of proboscis sensilla and their socket in males RPW by gamma irradiation, as a more careful step to understand the dynamic effect of rays on the chemical communication between insect-insect and insect-plant.

MATERIALS AND METHODS

Insects Red palm weevil adults were obtained from cocoons collected from infested date palm trees in the Menasheet El Keram and Tel Bani Tamim-Qalyubia-Egypt. The maintained pupae are used in laboratory of Atomic Energy Authority-Inshas-Central Labs-Kebab Building, according to the method described by (Mahmoud and Shoman, 2009). The pupae were transferred in cylindrical plastic dishes (15cm in depth x 37cm in diameter) with an amount of moist tissue fibers of sugarcane output from adult and larvae feeding. They were also kept at controlled laboratory conditions (27°C and 85% RH) and daily inspection was carried out until the adult emergency. The adult weevils were transferred into cylindrical glass jars (16cm in depth x 8 cm in diameter) with pieces of sugarcane as food, which was renewed almost every two or three days. Jars were covered with a network of metal wires.

Irradiated treatments From 25 to 30 RPW pupae (about 25 days) were kept in plastic containers (11cm in depth, 16 cm in diameter) and irradiated with 10, 15, 20 Gy of gamma radiation, using gamma cell-40 (cesium-137 irradiation unit), at the National Center for Radiation Research and Technology (NCRRT, Cairo).The one-day-old emerged males were separated and they fed on reeds. Three males were taken from each group. The dose rate was 0.758 rad/sec at the time of the present investigation. 55 Impact of Gamma Radiation on Male Proboscis of Rhynchophorous ferrugineus Scanning electron microscopy studies As for the examination of the proboscis of freshly killed normal and irradiated adult males, RPW of 5 old days were removed from the head cleaned in distilled water, immersed in 2.5% glutaraldehyde for 24 hours and then washed three times with 0.1 M phosphate buffered saline (PBS) (pH 7.4). The samples were fixed in osmic acid for 2 hrs and then underwent a series of ethanol concentrations (50, 70, 80, 90, 95, and 100%) for 15 mins for each (Kang et al., 2012). The proboscis was attached vertically to aluminum stubs and air-dried before getting coated with gold in a vacuum evaporator (Hazaa, 2010). The examination was conducted by using the Scanning Electron Microscope (SEM) (Joel JSM 5400) with an accelerating voltage of 10 kV.

Statistical analysis All data obtained were statistically analyzed and the variance ratios were calculated. The method of ANOVA is involved by using (SPSS) computer program, ver.15.0, and the significance among the samples was calculated at P ≤ 0.05.

RESULTS The head of red palm weevil is a prognathus type, bearing two large compound eyes as photoreception; there is one compound eye at each lateral side at the base of the proboscis of the head (Fig. 1). Optical dierentiation between male and female adults from the morphological feature of the proboscis and the hairbrush-like structures at the terminal 1/2 of the dorsal side for males, are absent in females. These brush hairs may be recognized as pheromone or mechanoreceptors (Fig. 1). There are different kinds of sensilla on this proboscis as follows:

Coeloconic sensilla (CI) Large number of CI is distributed on the lateral and frontal surface of the proboscis recorded as 176.7±25 (Fig. 2B and Table 1). Under high magnification, the sensilla appear to be formed of multiple straight tubules directed upward and giving a blunt shape, opening at the apex with many ridges on the surface wall of the sensilla whether they are broad or not. Each one arises from a narrow socket. These sensilla range from about 30.7 to 39.8 µm in length, and from about 53 to 63.6 µm in diameter at its base (Fig. 2C).

Coeloconic sensilla (CII) CII located in few numbers on the lateral surface of the proboscis recorded about one. The fingers are elongated and taper apically, with some tilt around the other, and appear to be directed downwards towards the basal margin of proboscis (Table 1). They emerge from a well-defined wide socket (finger-like). These sensilla range from about 18.9 to 21 µm in length, and are about 15.4 µm in diameter at its base (Fig. 2D). 56 MAHMOUD, E. A., GABARTY, A. Coeloconic sensilla (CIII) These sensilla found on the anterior part of the lateral and frontal surface of the proboscis recorded about 40.3±0.9 (Table 1). The finger-like processes of the sensilla are long and fused together and their tip is blunt. They extend with the same diameter and decrease apically in size. Most of the sensilla grow out from wide sockets with an amount of cuticular glands attached to some sensilla at bases and are more perpendicular on the antenna than other coeloconic sensilla. They outnumber the sensilla type CII and extend vertically on the proboscis surface at a different length. These sensilla range from about 15 to 26.9 µm in length, and from about 3.7 to 5 µm in diameter (Fig. 3A).

Coeloconic sensilla (IV) The sensilla IV are represented as 5 rows found on the frontal surface and as one or two rows around the trichiod sensilla on the lateral surface of the proboscis as about 62.3 (Figs. 2A, 3B, Table 1). These immovable hairs resemble sensilla CI but have various sizes. In addition, some of their tips are pointed and some are broad and long. They grow out from very narrow sockets where their bases of fingers occupy all cavities, attached to the margin of socket and are more perpendicular on the antenna than other sensilla. They appear pyramidal in shape, and their height gradually increases, ranging from about 31.4 to 93 µm, and about 71 µm in diameter as illustrated in Fig. 3B.

Squamiform sensilla (SI) A very few number of SI was found on the anterior lateral surface of proboscis, equal to about one sensilla (Fig. 2D, Table 1). The sensilla are smooth-walled hairs, arising from cuticular raised glands at its base with wide articulatory socket. Their height gradually and slightly increases, with a diameter at the base of about 1.9 µm. to be about 6.5 µm and grow less in size to about 4.2 µm at the tip. These sensilla are more curved from the base over the whole surface of the proboscis. They arise nearly from the center of the fine, broad-tipped, finger-like process at its end base, measuring about 6.5 μm in length and about 1.5 µm in diameter.

Trichoid sensilla (TrI, TrII) The sensilla TrI, TrII with different lengths are illustrated on the dorsal surface of proboscis. They are arranged clearly in two rows, with narrow spaces between them of approximately 192 μm, and combined the rows from the front, whereas the two sides remained as separated posterior (Figs. 2B, 4A). Each row is a set of rows ranging in number from three to four. The surface of sensilla appeared striated; they consist of fine long tubules that unite together (Fig. 3C). The sensilla seem vertical on the proboscis surface, near the base, forming an angle of about 45° and back again in a vertical-ascending in curvature; they are adherent to each other and dense. They stand aside from an articulatory and prominent socket with an amount of glands (Figs. 2B, 3C). They are long or short with blunt tipped sensilla. TrI were recorded at 57 Impact of Gamma Radiation on Male Proboscis of Rhynchophorous ferrugineus about 21.7±0.8 and ranged from 166 to 216 μm in length, 23 to 26 μm in diameter, whereas TrII were recorded about 160.3±1.5 and ranged from 63 to 83 μm in length, 15 to 21 μm in diameter (Table 1). The shortest sensilla are found in the frontal and lateral parts while the longest sensilla are behind them. They are short only in the distal row (Fig. 2B). From the same socket of TrI, TrII arise CV attached to their base (Fig. 5A).Some of these sensilla resemble the sensilla (CII) with some differences in size, about 76.1 μm in length and nearly 30.4 μm in diameter at their base (Fig. 3C). The other parts of CV are pyramidal in shape and are 26.6 μm in length and about 29.7 μm in diameter at their base (Fig. 5A).

Apodemes The surface of cuticle in RPW contains a large number of internal processes or bands (gab through the cuticle), which appear to be light or dark. Arcuate patterns seem to be superimposed in these bands. These bands contain a matrix of tinny protrusions (or filaments) on their internal margin that is internally oriented (Figs. 3B, D) and meet with the other filaments on the opposite side as funnel in shape. The protrusions appeared to be of an open type in the normal state (Fig. 3D). These bands are with different size and length along the cuticular surface between the different sensilla. Their orientation is in all direction at the anterior and lateral surface while most of them are in the dorsal surface between the two rows of Tr that are horizontal in direction (Figs. 2B, C).

Glands A number of glands could be illustrated on the anterior and lateral side of proboscis between CI, CII (Fig. 2D). However, they were found among CIII in few numbers. Most of them are circular in shape while few are rectangular and vary in their diameters (Figs. 2D, 3D).

The Effects of gamma radiation

Proboscis sensilla It is obvious that irradiation with dose 10 Gy had a slight effect on the features of the different types of sensilla as to with the normal state. Some CI, CIII changed in appearance and recorded 27.7±1.9, 5±0.6 (Table 1). The base of sensilla became less dense especially in the central part, but it become flattened at the apex, diffused and grew less dense and blunt. A number of tubules-like processes of CI and CIV became aggregated in two groups and their apical parts were ruptured (Fig. 4B). Also some CIII, cuticular glands attached to its bases are disappeared. Whereas the tubules of TrI, TrII seemed to be slightly separated on the terminal part and recorded about 8±0.6, 55±2.9 respectively (Fig. 4A). On the other hand, with the increase of radiation doses (15, 20 Gy), the changes in CI are as in dose 10 Gy but with an increase in number of malformations from about 58 MAHMOUD, E. A., GABARTY, A. 42.3±1.5 to 65.3±4.2 (Figs. 5B, 6B). As the sensilla CIII appeared to grow out from dipped sockets, small numbers of cuticular glands were attached to the base of sensilla and a percentage of malformations increased from about 9±0.6 to 13.3±0.8 (Figs. 5C, 6C). Additionally, in dose 15 Gy some of TrI, TrII were clearly and drastically affected where their tubules were completely separated and some of them were ruptured and recorded 15±0.5 and 93±4, respectively, (Fig. 5A). Most of TrI, TrII lost their normal curvature and adherence at the dose level 20 Gy and also recorded about 19±0.3, 137.7±4.6, respectively, in significant to control at (p≤0.05) (Fig. 6D, Table 1).

Apodemes The appearance of podemes differed due to the use of different doses of gamma radiation. The variances in size and depth were illustrated and increased with the increase of the doses (Figs. 4C, 5B, 6C). They appeared to be of an open type in the dose 10 Gy (Fig. 4C) and seemed to be few in the dose 15 Gy (Fig. 5C), whereas the majority of closed apodemes appeared in the use of doses 15 and 20 Gy (Figs. 5B, 6B, C).

Table 1. The deformity induced by gamma irradiation on the different types of male proboscis sensilla of R. ferrugineus.

Types of Sensilla Mean±SE Dose (Gy) CI CIl CIII CIV TrI TrII SI

Control 176.7±14.5 1 40.3±0.9 62.3 21.7±0.8 160.3±1.5 1

10 27.7±1.9* - 5±0.6* - 8±0.6* 55±2.9* -

15 42.3±1.5* - 9±0.6* - 15±0.5* 93±4* -

20 65.3±4.2* - 13.3±0.8* - 19±0.3* 137.7±4.6* -

The mean equals 3 replicate for each group-SE is the stander error-the mean difference is significant at the 0.05 level.

Fig. 1. Photograph of male head R. ferrugineus showing the sensilla at the dorsal side of proboscis. (antennae=An, proboscis=Pr, compound eyes=Co). 59 Impact of Gamma Radiation on Male Proboscis of Rhynchophorous ferrugineus

Fig. 2. (A) Types of sensilla located on the lateral view to normal proboscis. (B) Type I, II of trichoid sen- silla (Tr) located on dorsal view. (C) Type I of coeloconic sensilla (C) were distributed on the lateral and frontal surface. (D) Type II of coeloconic sensilla and squamiform sensilla (SI) were located in very few numbers on the anterior lateral surface (Apodemes=A and the arrows represented different size for glands).

Fig. 3. (A) Type III of coeloconic sensilla arising from wide sockets with amount of cuticular glands attached to its bases. (B) Type IV of coeloconic sensilla represented as 5 rows found on the frontal surface. (C) From the same socket of trichoid sensilla arise type CV of coeloconic sensilla attached to their base. (D) The opened apodemes contain matrix of tinny protrusions on its internal margin oriented perpendicular internally and the arrows represented different size for glands. 60 MAHMOUD, E. A., GABARTY, A.

Fig. 4. Irradiated R. ferrugineus with 10 Gy of gamma rays. (A) A number of tubules TrI, TrII seemed to be slightly separated on the terminal part. (B) The fingers of CI became aggregated in two groups and their apical parts were ruptured. (C) In most CIII, cuticular glands attached to its bases were disappeared. (D) Ruptured to some apical parts of tubules-like processes in CIV.

Fig. 5. Irradiated R. ferrugineus with 15 Gy of gamma rays. (A) Malformation on TrI, TrII. (B) The varying in sizes and deep of apodemes. (C) Irregular tubules of CI, CIII. 61 Impact of Gamma Radiation on Male Proboscis of Rhynchophorous ferrugineus

Fig. 6. Irradiated R. ferrugineus with 20 Gy of gamma rays. (A) shrinkage and atrophy of CIV. (B) Highly irregular sizes of closed apodemes. (C) increased malformations in CIII. (D) Most of TrI, TrII tubules were completely separated and some of them were ruptured and the arrow represented the gland.

DISCUSSION The obtained results indicated that there were eight different sensilla identified along the proboscis of male’s R. ferrugineus. All sensilla observed, in this study could be displayed external morphologies similar to those displayed by the previous studies performed on antenna (Mahmoud et al., 2011), and ovipositor of (Sharaby and Al-Dosary 2006, 2007) of R. ferrugineus. According to Baker and Ramaswamy (1990) the striations on the sensilla surface are regarded as one of the characteristic features of trichod sensilla. It has been suggested by Merivee et al. (1997) that asymmetries in the distribution pattern of olfactory sensilla on antennae might be due to the peculiarities of their behavior (waiting, walking, flying, antennal movement), which cause certain areas of the antennal surface to catch the wind-borne odour molecules more effectively than the others. These results are largely in conformity with those results reported in our study; the most numerous trichoid sensilla are arranged into two rows that exist on the dorsal surface of proboscis. Each row is a set of rows ranging in number from three to four. These sensilla are considered as pheromone receptors in the beetles Hylobius abietis (Mustaparta, 1973), and Psacothea hilaris (Castrejon-Gomez et al., 1999). The coeloconic-sensilla distributed along the proboscis of RPW resemble the sensilla recorded on the ants and wasps, whose behavior is guided by variations in temperature. Coeloconic sensilla are chemoreceptors that respond to air temperature changes (Rychty et al., 2009). Interestingly, the coeloconic sensilla are reported to 62 MAHMOUD, E. A., GABARTY, A. have receptor neurons that respond to host plants volatile compounds as described by Pophof (1997). In addition, very few mechanoreceptor squamiform sensilla were distributed on the anterior lateral surface of proboscis. The same sensilla elucidated on adults of rice water weevil, Lissorhoptrus oryzophilus with fluted surfaces, occurring on the escape, pedicel and funicular flagellum (Kang et al., 2012). Likewise, squamiform sensilla were found on elytra surface of Harmonia axyridis Pallas and was known as the micro-hair with a length of 8 μm, which was developed in the depression structure and located in the center (Li et al., 2012). The squamiform sensilla I, II were also found on the rostrum, antenna, elytra and tibia of Oryzophagus oryzae (Martins et al., 2012). In Hix et al. (2003) it was confirmed that its function might have to do with in mechanoreceptor and proprioreception. The microscopic observations of the males R. ferrugineus proboscis revealed a number of the glands most of which are circular in shape while few are rectangular. These glands defined by Chen et al. (2012) as glandular pores or glands on Dendroctonus valens antennal sensilla. Similar glands were also observed in Chilopod subtaxa as epidermal glands of differences size, named ‘flexo-canal epidermal glands (Müller et al., 2009). These glands have been thought to represent either a kind of lubricant for the antennae and their sensilla or appeasement glands or pheromone glands (Weis et al., 1999). In this work no significant changes could be observed by irradiation in the features of these glands in the treated males. Unfortunately we did not find any research performed on the effect of gamma ray on the insect’s proboscis but a lot of research papers have examined its impact on the antennae. In all insects, the rigidity of the exoskeleton is increased by four deep culicular invaginations, known as apodemes, which meet internally to form a brace for the exoskeleton and for the attachment of muscles (Chapman, 2003). The apodemes reported in RPW have different size and length along the cuticular surface between the different sensilla. The trend of the internal protrusions appeared inward (in funnel-shaped) with the doses of 15 and 20 Gy and this explains its function which may play an important role in regulating the evaporation of water from the body to overcome the rise in temperature. It also helps water balance to preserve its life where the sensitivity of the RPW to dry conditions was studied by Nirula (1956).These apodemes resemble to that illustrated in Drosophila where the muscle has detached from one of its apodemes. These effects show that the behavioral studies suggesting a dominant suppression are incomplete (Naimi et al., 2001). Some malformations were observed on proboscis sensilla of RPW male by gamma irradiation which increased with the increasing dose rays from 10, 15 to 20 Gy. However, only coeloconic sensilla IV were not affected by any dose. Irradiation with gamma rays on first generation ofSpodoptera littoralis antennal sensilla showed that the trichoid sensilla became low in number and also showed the loss of the central pegs from some coeloconic sensilla. With the increase of doses some spines of the coeloconic were knobbed, plus point, the central pegs were lost (El-Shall et al., 2004). 63 Impact of Gamma Radiation on Male Proboscis of Rhynchophorous ferrugineus Furthermore, Hazaa (2010) found that there are no malformations on squamiform or coeloconic sensilla by low doses of gamma rayes but the trichoid sensilla showed swelling of their base on S. littoralis or slight warping or that they twisted together with loss in number. With increase of radiation dose, the malformation of sensilla increased as the trichoid sensilla disoriented and collected forming bundles of sensilla and the terminal parts nodulations and the coeloconic shrank in many areas. At the high doses (150 Gy) the density of trichod sensilla became lower than in control at first generation to Galleria mellonella male (El-Kholy and Mikhaiel, 2008). Gharieb and El-Degwi (2002) recorded that the length and diameter of trichodea sensilla are affected by irradiation in Trogoderma granarium antenna. Hussien et al. (2001) stated that substerilizing doses of gamma radiation (5-12 Krad) reduced the density of trichoid sensilla and caused malformation to some of them in the antenna of the male moth of black cutworm, Agrotis ipsilon. The authors observed that the response of these irradiated males to sex pheromones was lower than in control males and its perception decreased with the increasing of the dose applied. These radiation effects were more pronounced in the parental generation than in F1 generation.

CONCLUSION This result identified eight different sensilla along the proboscis ofR. ferrugineus male. Most of the trichoid sensilla are either arranged into two rows or distributed with coeloconic and squamiform sensilla along the proboscis. Sensilla can play a role as mechanoreceptor, chemoreceptors and pheromone receptors. A large number of apodemes with different sizes and lengths was also observed on the integument. These apodemes may play an important role in regulating the evaporation of water from the body to overcome the rise in temperature and making water balance to preserve its life. The effect of the doses 10, 15 and 20 Gy of gamma radiation on the proboscis were tested. The percentage of malformations increased with the increase of dose, and this may affect feeding behavior and the insect’s plant interaction. Moreover, feeding behavior studies are recommended to detect the effect of these deformations on the host seeking behavior of irradiated males.

REFERENCES

Abraham, V. A., Al- Shuaibi, M. A., Faleiro, J. R., Abozuhairah, R. A., Vidyasagar, P. S. P. V. ,1998, An integrated management approach for red date palm weevil, Rhynchophorus ferrugineus Oliv. A key pest of date palm in the Middle East. Sultan Qabus University. Journal of Scientific Research, 3: 77-84. Baker, G. T., Ramaswamy, S. B., 1990, Tarsal and Ovipositor sensilla of Heliothis virescens and H. subflexa (Lepidoptera: Noctuidae). Proceedings of the Entomological Society of Washington, 92(3): 521-529. Castrejon-Gomez, V. R., Valdez-Carrasco, J., Cibrian-Tovar, J., Camino-Lavin, M., Rodolf Osorio, O., 1999, Morphology and distribution of the sense organs on the antennae of Copitarsia consueta (Lepidoptera: Noctuidae). Florida Entomologist, 82: 546-555. Chapman, R. F., 2003, The Insects: Structure and Function. 4th edn. Cambridge University Press. 64 MAHMOUD, E. A., GABARTY, A.

Chen, H. B., Zhang, Z., Wang, H. B., Kong, X. B., 2010, Antennal morphology and sensilla ultrastructure of Dendroctonus valens LeConte (Coleoptera: Curculionidae, Seolytinae), an invasive forest pest in China. Micron, 41: 735-741. De Bore, G., 2006, The role of the antennae and maxillary palps in mediating food preference by larvae of the tobacco hornworm Manduca sexta. Entomologia Experimentalis et Applicata, 119: 29-38. El-Kholy, E. M. S., Mikhaiel, A. A., 2008, Scanning electron microscopy on male antennae of the greater wax moth,Galleria mellonilla (L.) treated with gamma radiation. Isotope and Radiation Research, 40: 603-613. El-Shall, S. S. A., Shaurub, E.-S. H., Alm El-Din, M. M. S., 2004, Morphology of gamma irradiated antennal sensilla in the male moth of the cotton leafworm, Spodoptera littoralis (Boisd) (Lepidoptera: Noctuidae) using scanning electron microscopy. Arab Journal of Nuclear Sciences and Application, 37: 177-189. Faleiro, J. R., 2006, A review of the issues and management of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm during the last one hundred years. International Journal of Tropical Insect Science, 26(03): 135-154. Gao, Y., Luo, L. Z., Hammond, A., 2007, Antennal morphology, structure and sensilla distribution in Microplitis pallidipes (Hymenoptera: Braconidae). Micron, 38: 684-693. Gharieb, O. H., El-Degwi, M. S., 2002, Effect of gamma radiation on morphology of sensory structures for the antennae of Trogoderma granarium (Everts.) adults. The Journal of Agricultural Science, Mansoura University, 27: 2637-2654. Gunawardena, N. E., Kern, F., Janssen, E., Meegoda, C., Schafer, D., Vostrowsky, O., Bestmann, H. J., 1998, Host attractants for Rhynchophorus ferrugineus: identification, electrophysiological activity and laboratory bioassay. Journal of Chemical Ecology, 24: 425-437. Hazaa, M. A. M., 2010, Morphological ultrastructure, distribution and function of the antennal sensilla of the cotton leaf worm male moth as affected by gamma radiation and/or heat stress. Journal of Radiation Research and Applied Sciences, 3(4B): 1485-1508. Hix, R. L., Johnson, D. T., Bernhardt, J. L., 2003, Antennal sensory structures of Lissorhoptrus oryzophilus (Coleoptera:Curculionidae) with notes on aquatic adaptations. The Coleopterists Bulletin, 57: 85-94. Hussien, M. A., El-Nagar, S. E. M., Abd El-Fattah, H. M., 2001, Effect of gamma radiation on certain antennal sensilla of male Agrotis ipsilon (Hufn.) and its impact on sex pheromone perception. The Journal of Egyptian Academic Society for Environmental Development, 1(1): 139-148. Kang, G.-J., Zhu, Z.-R., Cheng, J.-A., Way, M. O., 2012, Antennal sensilla of parthenogenetic and bisexual Lissorhoptrus oryzophilus (Coleoptera: Curculionidae). Florida Entomologist, (95)1: 8-15. Li, X., Chi, D., Zhu, Y., Zhang, Z., Yu. J., 2012, The comparative study on adult surface ultrastructure of Harmonia axyridis Pallas (Coleoptera: Coccinellidae). African Journal of Agricultural Research, 7(43): 5779-5791. Liu, Y. S., 2006, The inducting and using of Dendrolimus superans (Lepidoptera: Lasiocampidae) to the volatiles of Larix gmelinii Disser. Northeast Forestry Univer. 1-60. (in Chinese) Lukmah, H., Alquat, S., 2002, Red palm weevil, approaching to integrated pest management. Ministry of Agriculture and Water. 174 pp. Mahmoud, E. A., Mohamed, H. F., El-Naggar, S. E. M., 2011, Ultrastructure alterations in the red palm weevil antennal sensilla induced by gamma irradiation. Isotope and Radition Research, 43(4): 1145-1161. Mahmoud, E. A., Shoman, A. A. , 2009, Histomorphological studies on the testes of the gamma irradiated red palm weevil, Rhynchophorus ferrugineus. Isotope and Radition Research, 41(3): 699-716. Martins, C. B. C., Almeidab, L. M., Zarbina, P. H. G., 2012, Scanning electron micrographs of Oryzophagus oryzae (Coleoptera, Curculionidae), plastron structure and swimming behavior. Micron, 43: 321-325. Merivee, E., Rahi, M., Luik, A.,1997, Distribution of olfactory and some other antennal sensilla in the male click beetle Agriotes obscurus L. (Coleoptera:Elateridae). International Journal of Insect Morphology and Embryology, 269(2): 75-83. 65 Impact of Gamma Radiation on Male Proboscis of Rhynchophorous ferrugineus

Müller, C. H. G., Jörg Rosenberg, J., Gero Hilken, G., 2009, Fine structure and phylogenetic significance of ‘flexo-canal epidermal glands’ in Chilopoda. Soil Organisms, 81: 269-294. Mustaparta, H., 1973, Olfactory sensilla on antennae of pine weevil, Hylobius abietis. Zeitschrift fur Zellforschung und Mikroskopische Anatomie, 144: 559-571. Naimi, B., Harrison, A., Cummins, M., Nongthomba, U., Clark, S., Canal, I., Ferrus, A., Sparrow, J. C., 2001, A tropomyosin-2 mutation suppresses a troponin I myopathy in Drosophila. Molecular Biology of the Cell, 12: 1529-1539. Nirula, K. K., 1956, Investigations on the pests of coconut palm. Part IV. Rhynchophorus ferrugineus F. Indian Coconut Journal, 9: 229-247. Onagbola, E. O., Meye, W. L., Boina, D. R., Stelinski, L. L., 2008, Morphological characterization of the antennal sensilla of the asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), with reference to their probable functions. Micron, 39: 1184-1191. Pophof, B., 1997, Olfactory responses record from sensilla coeloconica of the silkmoth Bombyx mori. Physiological Entomology, 22: 239-248. Rychty, M., Romani, R., Kuebler, L. S., Ruschioni, S., Roces, F., Isidoro, N., Kleineidam, C. J., 2009, The thermo-sensitive sensilla coeloconica of leaf-cutting ants (Atta volleneideri). Arthropod Structure and Development, 38: 195-205. Sharaby, A., Al-Dosary, M., 2006, Sense organs on the head of the red palm weevil adults Rhynchophorus ferrugineus Olive. (Coleoptera: Curculionidae) of Saudi Arabian strain. Bulletin of the Entomological Society of Egypt, 83: 99-132. Sharaby, A. M., Al-Dosary, M. M., 2007, Ultra morphological characteristics of sensory sensillae on the legs and external genitalia of the Red palm weevil Rhynchophorus ferrugineus (Oliv.). Saudi Journal of Biological Sciences, 14(1): 29-36. Skiri, H. T., Standen, M., Sandoz, J. C., Menzel, R., Mustaparta, H., 2005, Associative learning of plant odorants activating the same or different receptor neurons in the mothHeliothis virescens. Journal of Experimental Biology, 208: 787-796. Weis, A., Schonitzer, K., Melzer, R. R.,1999, Exocrine glands in the antennae of the carabid beetle, Platynus assimilis (Paykull) 1790 (Coleoptera, Carabidae, Pterostichinae). International Journal of Insect Morphology and Embryology, 28: 331-335.

Received: February 17, 2016 Accepted: February 07, 2017

J. Entomol. Res. Soc., 19(2): 67-71, 2017 ISSN:1302-0250

New Record of Neotrichoporoides (Hymenoptera: Eulophidae) from Georgia with some Taxonomic and Biogeographical Notes

George JAPOSHVILI1, 2* Viktor KOSTJUKOV3 Oksana KOSHELEVA3 Ekaterina YEGORENKOVA4

1Invertebrate Research Center, Agladze #26, Tbilisi-0119, GEORGIA. 2Institute of Entomology, Agricultural University of Georgia, Agmashenebeli Alley #240, Tbilisi, GEORGIA. 3All-Russian Research Institute of Biological Plant Protection, Krasnodar 350039, RUSSIA. 4Ulyanovsk State Pedagogical University, Ulyanovsk 431700, RUSSIA. e-mails: *[email protected], [email protected], [email protected], [email protected], [email protected]

ABSTRACT Here, two species of Neotrichoporoides Girault, 1913, are recorded from Georgia (in the Transcaucasia) for the first time: N. dispersus Graham and N. viridimaculatus (Fullaway). A difference to separate this genus from others in the Tetrastichinae is provided. A list of species in the genus in the Caucasus is given. Key words: Georgia, Tetrastichinae, Caucasus, Neotrichoporoides.

INTRODUCTION The Lagodekhi reserve was established in 1912. The Lagodekhi Protected Areas is one of the world’s best-preserved, primitive areas, with a diversity of natural landscapes and is located in the region of Lagodekhi, in the extreme North-Eastern part of the southern slopes of the Caucasus Mountains, the preserve extending from 590-3500 m. The Lagodekhi Protected Areas include both the Lagodekhi Nature Reserve (19749 ha) and the Managed Reserve (4702 ha) (Agency of Protected Areas, 2016). The genus Neotrichoporoides Girault (Hymenoptera: Chalcidoidea, Eulophidae) was erected by Girault (1913) based on the material collected in Australia, is a monotypic genus, Neotrichoporoides uniguttata Girault. Graham (1986) described N. mediterraneus and N. despersus from Spain and Madeira. Later Graham (1987) and Bouček (1988) synonymized many genera (=Aprostoceroloides Girault, 1913; Tetrastichomorpha Girault, 1913; Trichaporoidella Girault, 1913; Paraprostocetus Girault, 1915; Burksia Fullaway, 1955; Dubiostalon Szelenyi, 1981; Neogaleopsomyia Narendran, 2005) with Neotrichoporoides Girault and updated the number of species assigned to the genus. This large genus has a wide distribution, and is especially species rich in Africa, Asia, and Australia (Graham, 1987). According to Noyes (2016) 68 JAPOSHVILI, G., KOSTJUKOV, V., KOSHELEVA, O., YEGORENKOVA, E. 19 species are recorded from Europe. Species belonging to the genus are parasitoids of Muscidae and Diopsidae (Diptera) on coarse grasses (Poaceae). Few specimens of Neotrichoporoides have been collected; Graham (1987) in his revision, found only 139 females and 48 males in European museums and collections. Types of eight species are described in the Graham’s monography (Graham, 1987), coming from a study of 41 females and 14 males. The most well collected species in European museums was N. dispersus Graham, with 37 females and 13 males. Most specimens of N. biogradensis Graham (5 females and 3 males) were collected by Bouček during a single week (13-19.07.1968) in Dalmatia, Biograd na moru, Croatia. In 2003, in plum orchards in Prietokskiy, Georgievskiy district of Stavropolskiy Krai (Russia), sweeping netting from mid-July to mid-August by Kostjukov, Kosheleva and Khomchenko recovered 129 females and 26 males of following species: Neotrichoporoides szelenyii (Erdös), N. viridimaculatus (Fullaway), N. cavigena Graham, N. disperses Graham, and N. mediterraneus Graham (Kostjukov et al., 2004). Malaise traps set in the same area from June to October (2013), however, failed to capture any Neotrichoporoides species. This study represents part of the material collected in Lagodekhi reserve, using malaise traps during the whole growing season of 2014, to study biodiversity of Hymenopterans in Lagodekhi protected areas.

MATERIAL AND METHODS To investigate the Hymenoptera biodiversity of the Lagodekhi protected areas in George, Malaise traps were used during the entire growing season of 2014 in a range of habitats over an altitudinal gradient. Malaise traps (one per site) were set in: (1) low altitude forest (450-750 m), (2) middle altitude forest (750-1250 m), (3) high altitude forest (1250-1800 m), (4) subalpine forest (1800-2000 m), (5) subalpine fields and shrublands (2000-2500 m), and (6) the alpine zone (>2500 m). Collecting began on 02.04.2014 and lasted until 07.11.2014, although in alpine and subalpine areas collecting was started later (subalpine 05.05.2014; alpine 23.05.2014) and completed earlier (06.10.2014), due to climate conditions at those altitudes. Collected material was retrieved every 10 (± 2) days and placed at first in 96% Ethanol, and later sorted, dried, mounted and labeled according to methods described by Noyes (2016). Identification was done by the second, third and fourth authors, using modern keys (Graham, 1987) and papers giving original descriptions (Kostjukov, 2004; Yegorenkova and Kostjukov, 2006), and the collections of the Zoological Institute of the Russian Academy of Sciences (St. Petersburg) and All-Russian Research Institute of Biological Plant Protection (Krasnodar). Malaise traps were obtained from BandN Entomological services (http://www. entomology.org.uk/). Information about the biology and distribution of species captured is given in the Universal Chalcidoidea Database (Noyes, 2016). All voucher specimens were deposited in the Entomological collection of Agricultural University of Georgia, Tbilisi, Georgia (IEAUG). 69 New Record of Neotrichoporoides from Georgia RESULTS

Diagnosis for genus Neotrichoporoides:

Female: Length: 1.4-3.3 mm. Thorax weakly arched 1.6-2.1x as long as broad. Pronotum conical, at least about half as long as mesoscutum. Scutellum usually at least as long as broad and nearly or almost as long as mesoscutum, occasionally a little broader than long or distinctly shorter than mesoscutum. Propodeum medially at least very slightly, but often much longer than dorsellum; callus with 3-7 setae. Forewing 2.5-2.95x as long as broad, with costal cell narrow; SM (Submarginal vein) with 3-8 dorsal setae; M (Marginal vein) 5.5-9.5x length of ST (Stigmal vein), the latter very short. Antenna with 4 anelli, funicular segments usually moderately to strongly elongate (2.8-5.5x as long as broad), rarely short (1.2-1.5x as long as broad). Sensilla very numerous in 3-5 rows on each funicular segments. Fovae of malar sulcus small or moderate, extending 0.33-0.75 length of malar space. Body usually with distinct metallic tints on dark parts, but some predominantly or completely yellow species without metallic tints.

Male: Length: 1.2-2.8 mm. Differs from female as follows. Antennae with ventral plaque of scape usually extending most of length of scape, occasionally short and placed in upper or lower half, or in the middle; funicle with 4 segments; segments of clava, especially C1, tending to be separated by strong constriction. Differential diagnosis between Neotrichophoroides and other Tetrastichinae is given in the table 1.

Species list of Neotrichoporoides from Lagodekhi reserve (Georgia)

Neotrichoporoides dispersus Graham, 1986

Material examined: Lagdekhi reserve, Mt. Kudigora, 41˚ 54.371’ N, 46˚ 20.004’ E, 2558 m alt, malaise trap, 26.07.2014-5.08.2014, 1♀, G. Japoshvili and G. Kirkitadze. Distribution: Croatia, Italy, Moldova, Portugal (Madeira), Russia (Stavropolski Krai), Serbia, Spain (Canary Islands) (Graham, 1987, Kostjukov et al., 2004, Noyes, 2016).

Neotrichoporoides viridimaculatus (Fullaway, 1955)

Material examined: Lagdekhi reserve, Mt. Kudigora, 41˚ 51.149’ N, 46˚ 17.266’ E, 666 m alt, malaise trap, 23.04.2014-03.05.2014, 1♀, G. Japoshvili and G. Kirkitadze. Distribution: Argentina, Bermuda, Bulgaria, Caribbean, Colombia, Cuba, Czech Republic, France, Hawaii, Hungary, India, Italy, Portugal (incl. Madeira), Russia 70 JAPOSHVILI, G., KOSTJUKOV, V., KOSHELEVA, O., YEGORENKOVA, E. (Krasnodarskii Krai, Stavropolskiy Krai, Karachai-Cherkess AR), Slovakia, Sweden, Turkey, USA (Graham, 1987; Kostjukov et al., 2004; Noyes, 2015). Table 1. Differential diagnosis of Neotrichoporoides and other Tetrastichinae. The species of genus Neotrichoporoides The other species of subfamily Tetrastichinae Female Female 1. Length 1.4-3.3mm 1. Length 0.5-4.5mm 2. Pronotum conical, at least about half as long as mesoscutum. 2. Pronotum not conical much shorter than 0.5 length of mesoscutum; if pronotum conical then fore tibia with 2 spur (Crataepus) or SM with 2 dorsal setae (some Ootetrastichus) or funicular segments quadrate (Syntomosphirum) or mid lobe of mesoscutum with numerous setae (Melittobia). 3. Antenna with funicular segments usually moderately to strongly 3. Antenna with funicular segments transverse, quadrate or 1.1- elongate (2.8-5.5x as long as broad) with sensillae very numerous 2.5x as long as broad, with sensillae in 1-2 rows on each funicular in 3-5 rows on each funicular segments. segments; if funicular segments moderately or strongly elongate, then funicle with 4 segments (Hyperteles) or SM with 2 dorsal setae (some Ootetrastycus) or pronotum not conical, scutellum broader than long and distinctly shorter than mesoscutum (Kolopterna and some Aprostocetus). Anterior margin of clypeus distinctly bidentate. 4. SM of forewing with 3-8 dorsal setae. 4. SM of forewing with 1-2 dorsal setae; if with 3-8 dorsal setae then pronotum not conical or funicular segments not moderately to strongly elongate or funicle with 4 segments. Male Male 1. Length 1.2-2.8 mm 1. Length 0.5-4.0mm 2. Antenna with segments of clava tending to be separated by 2. Antenna with segments of clava not separated by strong strong constriction. constrictions, often not distinctly segmented, 2 segmented or solid; if clava segments separated by strong constrictions then malar sulcus with fovea below the eye (Hyperteles, Kolopterna, Ootetrastichus, some Aprostocetus). 3. Hosts Muscidae and Diopsidae (Diptera) species on coarse 3. Hosts Cecidomyiidae (Diptera) species on coarse grasses grasses (Gramineae). (Gramineae), other insects, Eriophyidae (Arachnida) and gall forming Nematodes (Nematoda).

DISCUSSION These recoveries of specimens within the genus Neotrichoporoides are a first record for Georgia; both species found in the Lagodekhi reserve are new for the fauna of Trancaucasia. Only one species N. szelenyii (Erdos, 1951) has been recorded from Azerbaijan (1♀, Baku, 02.07.1967, Bouček) before our study (Graham, 1987). Collections have been made in several locations: (1) The genus Neotrichoporoides is most diverse in protected areas of the greater Caucasus Mts. Five species (N. cavigena Graham, 1987, N. dispersus Graham, 1986, N. mediterraneus Graham, 1986, N. szelenyii (Erdos, 1951), and N. viridimaculatus (Fullaway, 1955)), have been recorded from around the health resort of Caucasus Mineralnye Vody (Georgievsk, Stavropolskiy Krai, Russia) (Kostjukov et al., 2004). (2) Four species, N. dispersus, N. mediterraneus, N. viridimaculatus, and N. trjapitzini Kostjukov, 2004 (Kostjukov, 2004) were collected from the surroundings of city of Sochi. (3) In the territory of the All-Russian Research Institute of Biological Plant Protection (Krasnodar, Russia), four species (N. cavigena, N. mediterranea, N. szelenyii and N. viridimaculatus) have been recorded by the authors and (4) In the reserves of Tersko-Kumskoe Sands (Dagestan, Russia), four species were recorded: N. dispersus, N. mediterraneus, N. szelenyii and 71 New Record of Neotrichoporoides from Georgia N. viridimaculatus (Gunasheva et al., 2015). Thus in the Caucasus region, 6 species have been recorded.

ACKNOWLEDGEMENTS We would like to thank to Dr. Roy Van Driesch (of VanDriescheScientificEditing. com), Amherst, Massachusetts, USA, for his kind help improving English of the presented article and for his valuable comments. We also thank the Rustaveli National Science Foundation for financial support under ref: FR/221/7-110/13. Finally we express our gratitude to Mr. Meri Salakaia and Mr. Marine Batsankalashvili for their kind help in sorting material.

REFERENCES

Agency of Protected Areas, 2016, http://apa.gov.ge/en/. 08.01.2016. Bouček, Z., 1988, Australasian Chalcidoidea (Hymenoptera). A biosystematic revision of genera of fourteen families, with a reclassification of species. CAB International, Wallingford, Oxon, U.K., Cambrian News Ltd; Aberystwyth, Wales, 832. Girault, A. A., 1913, New genera and species of Chalcidoid Hymenoptera from North Queensland. Archiv für Naturgeschichte, 79: 46-51 Graham, M. W. R. de V., 1986, Four new species of Eulophidae (Insecta, Hymenoptera) from Madeira and Europe. Bocagiana, 95: 1-9. Graham, M. W. R. de V., 1987, A reclassification of the European Tetrastichinae (Hymenoptera: Eulophidae), with a revision of certain genera. Bulletin of the British Museum, 55(1): 1-392. Gunasheva, Z. M., Kostjukov, V. V., Kosheleva O. V., 2015. Comparative analysis of fauna Eulophidae from inner Dagestan and Tersko-Kumskie sands. Herald of Dagestan Scientific Centre, 57: 9-12. Kostjukov, V. V., 2004, New species of the genus Neotrichoporoides Girault, 1913 (Hymenoptera, Eulophidae) from Algeria, Russia and Tirkmenistan. Biological Plant Protection as a Basis for Stabilization of Agroecosystem, 2: 159-170. Kostjukov, V. V., Kosheleva, O. V., Khomchenko, E. V., 2004, Species of the genus Neotrichoporoides Girault, 1913 (Hymenoptera: Eulophidae) recorded for the first time from Russian fauna. Biological Plant Protection as a Basis for stabilization of Agroecosystem, 2: 181-184. Noyes, J. S., 2016, http://www.nhm.ac.uk/chalcidoids. 18.02.2016. Yegorenkova, E. N., Kostjukov, V. V., 2006, New species of the genus Neotrichoporoides Girault, 1913 (Hymenoptera: Eulophidae: Tetrastichinae) from Middle Volga Region of Russia. Russian Entomological Journal, 15(4): 421–422.

Received: February 18, 2016 Accepted: June 23, 2017

J. Entomol. Res. Soc., 19(2): 73-82, 2017 ISSN:1302-0250

Diversity and Nesting Preferences of Camponotus lateralis Group Species on Western Balkan Peninsula (Hymenoptera: Formicidae)

Adi VESNIC1* Rifat SKRIJELJ1 Sadbera TROZIC-BOROVAC1 Zeljko TOMANOVIC2

1Biology department, Faculty of Science, University of Sarajevo, Zmaja od Bosne 33-35, 71000 Sarajevo, BOSNIA AND HERZEGOVINA e-mails: *[email protected], [email protected], [email protected] 2Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16, Belgrade, SERBIA, e-mail: [email protected]

ABSTRACT On western Balkan Peninsula Camponotus lateralis group Emery, 1925 is represented by three species: Camponotus lateralis (Olivier, 1791), C. piceus (Leach, 1825) and C. dalmaticus (Nylander, 1849). Camponotus lateralis group is founded by ecologically subordinated ants that are dominantly adapted to habitats in Mediterranean region. In contrast to Camponotus lateralis and C. dalmaticus, C. piceus is also found in the Dinaric region on open hot meadows. In microhabitat level, Camponotus piceus is building colonies in ground and open thermophilic Mediterranean and Dinaric grassland habitats. Camponotus lateralis and C. dalmaticus are associated with woodland and shrub land habitats in the Mediterranean region. Camponotus lateralis construct colonies in dry wood, however 11% of colony findings were under stone. Similar to Camponotus lateralis, Camponotus dalmaticus build nests in rotten wood parts, roots and tree trunks that were underground. Collected field data indicate that overlap in colony construction niche between analysed species is low due to different habitat and nesting site preferences. Key words: Camponotus, Myrmentoma, ants, nesting preferences, Western Balkan Peninsula.

INTRODUCTION Ants of Camponotus lateralis group Emery, 1925 belong to the subgenus Myrmentoma Forel, 1912. Morphologically Camponotus lateralis group species differ from other groups within the subgenus Myrmentoma by flattened dorsal surface of the propodeum. The posterior propodeum can be curved in or flat and the dorsal surface of the thorax is either with or without mesopropodeal depression (Radchenko, 1997). Biosystematic data indicate that the diversity of the Camponotus lateralis group on western Balkan Peninsulais is larger than expected: two undescribed species provisionally named by Seifert (2007) as Camponotus lateralis sp. 2 and C. piceus sp. 2, Seifert (2007) were reported for the investigated area. According to a recent unpublished investigation of Seifert, C. lateralis sp. 2 is still a discrete intraspecific morph of C. lateralis meanwhile the heterospecificity of C. piceus sp. 2 from Camponotus piceus could be clearly confirmed (Seifert pers. comm.). 74 VESNIC, A., SKRIJELJ, R., TROZIC-BOROVAC, S., TOMANOVIC, Z. Camponotus honaziensis Karaman and Aktaç, 2013 has similarities with Camponotus lateralis sp. 2. It is possible that Camponotus honaziensis from Montenegro; reported by Bračko et al. (2014); actually represents Camponotus lateralis sp. 2. According to Borowiec and Salata (2013) Camponotus honaziensis is equal to Camponotus lateralis sp. 2 and it is widely distributed in Mediterranean areas. Camponotus lateralis group species are a common element in the ant fauna of the Mediterranean region, but it is still a less known component of woodland and grassland habitats in the western Balkan Peninsula due to their subordinated position in ant communities and cryptic nests. Aside from faunistical investigations, data regarding species autecology and general biology of Camponotus lateralis group are scarce (Müller, 1923; Zimmermann, 1934; Kaudewitz, 1955; Seifert, 2007). With this in mind, the primary goal of the current study was to investigate diversity of Camponotus lateralis group in the western Balkan Peninsula and to gather general zoological field data which would improve the understanding of autecology of Camponotus lateralis group. To determine habitat structure parameters that are important for nesting aggregation and habitat preference of Camponotus lateralis group on western Balkan peninsula we investigated number of colonies in regard to their geographical region, habitat type, habitat structure and nesting site microhabitat preferences.

MATERIAL AND METHODS The researched area of western Balkan Peninsula included: Montenegro, Bosnia-Herzegovina and Croatia. In the areas above we investigated presence of Camponotus lateralis group species within three regions: Mediterranean, Dinaric Alps and Continental region. The field investigations were conducted from May to September between 2013 and 2015.

Investigated sites In Montenegro the investigated areas were: 1-Herceg Novi, Savinjska wood (lat. 42.453°; lon. 18.552°). In Croatia: 2-Konavle, Lovorno: (lat. 42.547°; E18.361°), 3-Dubrovnik, Velika and Mala Petka: (lat. 42.650°; lon. 18.071°), 4-Peljesac Peninsula, the municipality Janjina (lat. 42.923°; lon. 17.427°), 5-Split (lat. 43.511°; lon. 16.413°) and 6-Pula (lat. 44.881°; lon. 13.881°). In Bosnia-Herzegovina field investigations were conducted in the Mediterranean part, 7-Neum (lat. 42.924; lon. 17.616), 8-Blagaj (lat. 43.257°; lon. 17.898°) and in the Dinaric areas: 9-Velez (lat. 43.348°; lon 17.895°), 10-Diva Grabovica (lat. 43.596°; lon. 17.712°), 11-Sarajevo (lat. 43.858°; lon. 18.447°), 12-Visegrad (lat. 43.773°; lon.19.274°), 13-Zavidovici (lat. 44.424°; lon. 18.177°), 14-Sanski Most (lat. 44.767°; lon. 16.668°), 15-Bihac (lat. 44.812°; lon. 15.870°), 16-Martin Brod (lat. 44.502°; lon. 16.156°), 17-Drvar (lat. 44.379°; lon. 16.384°). In Continental region of Bosnia and Herzegovina we examined three localities: 18-Orasje (lat. 45.028°; lon. 18.717°), 19-Brcko (lat. 44.863°; lon. 18.831°) and 20-Janja (lat. 44.663°; lon. 19.258°) (Fig. 1). 75 Diversity and Nesting Preferences of Camponotus lateralis Studied habitats Mediterranean forest habitats were represented with Quercetalia ilicis and Pinion heldreichi forest. Mediterranean scrub lands habitats were represented with: Ostryo-Quercetum illicis and Erico-Cistetalia habitats. Mediterranean grassland habitats were represented with: Thero-Brachypodietalia, Scorzoneratalia villosae meadows. Dinaric forest and scrub lands habitats were represented by: Pinetum sylvestris dinaricum, Ostryo-Fagetum, Carpinion betuli. Dinaric grassland habitats: Arrenateretea, Brometalia erecti, Calluno-Festucion capillatae, Festuco-Brometalia, Festucion vallesiacae. Continental forest and scrub lands habitats were represented by: Quercetum patraeae-cerris pannonicum, Quercetalia pubescentis. Continental grassland habitats: Molinion caeruleae, Deschampsietum cespitosae.

Sampling methods On 20 locations and 61 sub locations we have applied the active sampling method. On each sub locality we investigated on three sites that were recognized as a forest, a scrub land and a grassland habitats type. Each of the habitat types was actively sampled on10 plots of 7X7 meters and no more than 5 meters between plots. In total 1830 plots were analyzed. Sampled ants were stored in 96% ethanol and identified using identification keys (Agosti and Collingwood, 1987; Seifert, 2007) then deposited in the Laboratory for Animal ecology at the Faculty of Science in Sarajevo. All samples were labelled and cross referenced with field protocol numbers.

Habitat structure analysis The field protocol included information on sample code, municipality, locality, date, GPS coordinate, altitude, slope, exposition, region (Mediterranean, Dinaric and Continental) and the level of habitat degradation (forest, scrub land and grassland/ ruderal). Ant colony characteristics are: nest type, placement of the nest and height of the nest above ground. Analysis of forests sublevels included: percentage of the stone coverage and stone diameter, moss height and relative coverage, perennial plant level height and relative coverage, bush level height and relative coverage. Upper storey, tree level height, relative coverage and average diameter breast height were included in the protocol. The field protocol was applied to record information on newly found colonies and on the scouting workers. Transect line intersect method: a 100 meters in length was used for the assessment of the relative abundance of habitat structure parameters. The transect line was either placed in the center of the colony or where workers were found. Along a transect the newly found workers were considered as individuals from the same colony if find on 7x7 plots. In case of finding a new colony in the transect new data were collected on a separate protocol. Analysed structural parameters 76 VESNIC, A., SKRIJELJ, R., TROZIC-BOROVAC, S., TOMANOVIC, Z. were measured five times within the transect (starting point, 25, 50, 75, and 100 m of the transect).

Specimens identification Camponotus lateralis group species were identified using taxonomical keys Agosti and Collingwood (1987), Seifert (2007) and for Camponotus honazienzis we used original description according to Karaman and Aktaç (2013). Camponotus piceus sp. 2 and Camponotus lateralis sp. 2 morphs were identified by morphometric characters according to Seifert (2007). Continuous descriptive data of habitat structures were analysed by descriptive statistics, ANOVA and post hoc Newman-Keuls test. Connection between habitat structural parameters and number of ant colony findings was tested by linear regression.

RESULTS Field investigations confirmed that three ant species from Camponotus lateralis group are present in the investigated area: Camponotus lateralis (Olivier, 1791), C. dalmaticus (Nylander, 1849) and C. piceus (Leach, 1825). The distinct hairy morph of Camponotus lateralis (“Camponotus lateralis sp. 2” of Seifert, 2007) was collected from one colony in Montenegro, Herceg Novi (Savinjska wood), second finding were workers sampled in May 2013 from Neum. Morph of Camponotus piceus (“Camponotus piceus sp. 2” of Seifert, 2007) was collected from Bosnia-Herzegovina: Mostar (Diva Grabovica), Sarajevo (Dariva) and Croatia: Konavle (road from Lovorno to Kuna Konavoska) (Fig. 1). On the investigated area we found and collected samples from 10 colonies of Camponotus piceus sp. 2. In the investigated areas we have collected 128 colonies: Camponotus dalmaticus (17.2% sampled colonies), Camponotus lateralis (38.3%) and Camponotus piceus (44.5%). Most of the Camponotus lateralis group samples were collected in the Mediterranean region, 78.3%. In the Mediterranean region we collected 93.4% of Camponotus dalmaticus, 94.4% of Camponotus lateralis and 59.1% of Camponotus piceus colonies. Due to unequal intensity of sampling between different regions and habitat types, relative abundance of Camponotus lateralis group findings was separately computed for each analysed category (Table 1). In the Mediterranean region of 1.050 plots the total number of sampled colonies was 104 or 9.9%. In the Dinaric region we investigated 660 plots and the total number of positive plots was 24 or (3.6%) (Table 1). Relative number of colonies shows that Camponotus lateralis and C. dalmaticus are more common in the Mediterranean regions, while C. piceus has similar abundancy in the Mediterranean and the Dinaric regions (Table 1). On 120 plots in the Continental region we did not detect Camponotus lateralis group species. 77 Diversity and Nesting Preferences of Camponotus lateralis

Fig. 1. Camponotus lateralis group distribution map on western Balkan peninsula: The circle with a number indicates locations: 1-Herceg Novi and Savinjska wood, 2-Konavle, Lovorno, 3-Dubrovnik, Velika and Mala Petka, 4-Peljesac, Janjina, 5-Split, 6-Pula, 7-Neum, 8-Blagaj, 9-Velez, 10-Diva Grabovica, 11-Sarajevo, 12-Visegrad, 13-Zavidovici, 14-Sanski Most, 15-Bihac, 16-Martin Brod, 17-Drvar, 18-Orasje, 19-Brcko, 20-Janja; letters: L-Camponotus lateralis, L2-C. lateralis sp. 2, D-C. dalmaticus, P.-C. piceus, P2-Camponotus piceus sp. 2. Table 1. Absolute number and percentages of tested plots with Camponotus lateralis group colonies in the western Balkan Peninsula.

Region Mediterranean Dinaric

Total number of testing sites = 1830 1050 660

Camponotus lateralis (47) 0.044 (2) 0.30

Camponotus dalmaticus (21) 0.020 (1) 0.10

Camponotus piceus (36) 0.034 (21) 3.20

Relative distribution of Camponotus lateralis group species in the Mediterranean and the Dinaric region was analysed in order to determine nesting preferences toward habitat type. In the Mediterranean forest habitats colonies of Camponotus lateralis and C. dalmaticus had the highest abundance. Colonies of Camponotus piceus were dominantly built in the Mediterranean grassland habitats (Table 2). 78 VESNIC, A., SKRIJELJ, R., TROZIC-BOROVAC, S., TOMANOVIC, Z.

Table 2. Absolute number and percentages of positive testing surfaces with Camponotus lateralis group colonies: M-mediterranean, D-dinaric, C-continental, f-forest habitat, s-scrub land habitat, g-grass- land habitat.

Region M f M s M g D f D s D g

Total number of testing sites = 1830 350 350 350 220 220 220

Camponotus lateralis 9.4 3.4 0.5 0.0 0.0 0.9

Camponotus dalmaticus 3.7 2.3 0.0 0.0 0.0 0.5

Camponotus piceus 0.6 3.7 6.0 1.8 2.3 5.5

Absolute frequency

Camponotus lateralis 33 12 2 0 0 2

Camponotus dalmaticus 13 8 0 0 0 1

Camponotus piceus 2 13 21 4 5 12

The findings ofCamponotus lateralis group were concentrated in the range of 0-200 meters above sea level. Within the range of 0-200 meter altitude we collected 91.8% (45/49) samples of Camponotus lateralis, 95.5% (21/22) samples of Camponotus dalmaticus and 26.3% (15/57) samples of Camponotus piceus (Table 3). Table 3. Absolute number and percentages of findings of Camponotus lateralis group species in relation to altitude.

Altitude 0-100 100-200 200-300 300-400 400-500 500-600 over 600

Number of plots 690 360 360 60 150 150 60

Camponotus lateralis 3.9 5.0 0.6 1.7 0.0 0.7 3.9

Camponotus dalmaticus 1.0 1.4 1.9 5.0 0.0 0.0 1.0

Camponotus piceus 2.6 4.4 1.7 15.0 0.0 4.7 2.6

Absolute frequency

Camponotus lateralis 27 18 2 1 0 1 0

Camponotus dalmaticus 7 5 7 3 0 0 0

Camponotus piceus 18 16 6 9 0 7 1

Due to positively skewed data beside arithmetic, the geometric mean and median were calculated (Table 4). Colony construction site preferences in the investigated group show that Camponotus piceus build colonies dominantly in ground which represents 84.2% of findings and 15.8% in ground under rocks.Camponotus lateralis build colonies dominantly in dry dead tree trunks (65.3% of colonies) (Table 5). All of the Camponotus lateralis colonies were found either in dry hard dead wood trunks or in dry trees branches. The number of Camponotus lateralis colonies built under stone was 6.1% (Table 5). The colonies of Camponotus lateralis that were under rocks were a combination of wood and soil in a sort of galleries extending in the ground. However, one colony of Camponotus lateralis was built in a concrete wall. 79 Diversity and Nesting Preferences of Camponotus lateralis

Table 4. Camponotus lateralis group species average elevation above sea level preferences in Western Balkan Peninsula. Average ± standard deviation Structural parameter in habitat Species (n=number of cases) Geometric mean – Median (minimal-maximal value) 176.6±112.0 C. dalmaticus (n=22) 128.8 – 191.5 (15.0, 376.0) 111.9±96.8 Altitude (m) C. lateralis (n=49) 85.7 – 81.0 (16.0, 568.0) 233.2±195,5 C. piceus (n=57) 168.2 – 198.0 (14.0, 725.0) Height of colonies built in tree trunks and branches in Camponotus lateralis group was analysed. Colonies of Camponotus lateralis were 0-264 cm higher over ground, mode = 20. In Camponotus dalmaticus colonies built in tree logs and branches were at height 0-20 cm, mode = 5. Table 5. Absolute and relative numbers of findings of colonies regarding place of colony construction; absolute number (A) and percentages (P) of findings.

Dry log Rotten log Dry branch on tree Under bark In ground Under rock

A 32 4 8 2 0 3 Camponotus lateralis P 65.3% 8.2% 16.3% 4.1% 0.0% 6.1%

A 2 13 2 0 0 5 Camponotus dalmaticus P 9.1% 59.1% 9.1% 0.0% 0.0% 22.7%

A 0 0 0 0 48 9 Camponotus piceus P 0.0% 0.0% 0.0% 0.0% 84.2% 15.8%

All three species are building colonies dominantly in habitats in south and south-east exposition; Camponotus piceus 53.5%, C. lateralis 51.8% and C. dalmaticus 22.7% of detected colonies. The average ground slope in habitats of Camponotus lateralis was in range 0°- 60°, mode = 5°, in Camponotus dalmaticus 0°- 40°, mode = 10° and in Camponotus piceus 0°- 45°, mode = 15°. Analysis of structural parameters in habitats where colonies of Camponotus lateralis group were constructed indicated that all three species are building colonies on moderately rocky substrates. Most of the colonies were found in habitats with 10%-40% of surface covered with stones; in Camponotus lateralis 73.1% of colony findings, C. piceus 73.7% and C. dalmaticus 81.9%. Trees level shows most consistency and clearly indicates a differentiation between analysed Camponotus lateralis species. Linear regression analysis indicated that Camponotus piceus has a negative linear correlation with trees level coverage y = -0.3014x + 24.571, R² = 0.5258, p < 0,00, while Camponotus lateralis and C. dalmaticus show positive regression. Statistically significant difference by ANOVA and post hoc Newman-Keuls test at (p<5%)in tree diameter was detected between Camponotus lateralis and C. piceus habitats. 80 VESNIC, A., SKRIJELJ, R., TROZIC-BOROVAC, S., TOMANOVIC, Z. CONCLUSIONS AND DISCUSSION In western Balkan Peninsula, we have confirmed the presence of Camponotus lateralis sp. 2, and C. piceus sp. 2 morphs. Confirmed morphs had sympatric distribution with Camponotus lateralis and C. piceus. Camponotus lateralis sp. 2 was represented by colony from Herceg Novi (Savina wood) and samples of Camponotus lateralis sp. 2 workers from Neum are representing the northern most findings. Nesting preferences of Camponotus lateralis group species can be explained by ants’ temperature preferences. The ant nest temperature can significantly affect the development time of immature stages (Porter, 1988, Heinrich, 1993). Cold climate ants are building ant hills over the ground and construct colonies on sites with higher insolation (Brian and Brian, 1951; Pontin, 1960; Lach et al., 2010). Temperate forests ants as Camponotus ligniperdus and C. herculeaneus build colonies usually in wood. Wood is a good insulator and prevents rapid temperature changes in colonies. North temperate ant species also occupy dead logs and stumps that are heating more rapidly below bark or in surface galleries (Hölldobler and Wilson, 1990). According to our findingsCamponotus lateralis and Camponotus dalmaticus are thermophiles species with narrower niche in compare to Camponotus piceus. Colonies of Camponotus lateralis and C. dalmaticus are aggregated in the Mediterranean habitats on lower elevations Colwell et al. (2008) which indicate the thermophilic nature. In the Mediterranean regions, the main differences between Camponotus lateralis group species were in their habitats preferences. In the Mediterranean regions Camponotus piceus was building colonies in open grassland habitats while Camponotus lateralis and C. dalmaticus were building in forest and scrub land habitats. Colonies of Camponotus lateralis were found in habitats with largest tree diameters which indicates a preference toward old forests. Compared to other Camponotus lateralis group species, Camponotus lateralis shows a variety of micro habitats for colony construction and had the broadest ecological niche on western Balkan Peninsula. In contrast to our investigations Zimmermann (1934) has discovered nests of Camponotus lateralis in south Dalmatia but only in ground. Our data do not indicate regional preferences towards soil as nesting place for Camponotus lateralis or local intraspecific differences in the investigated area. The main detected difference in colony construction betweenCamponotus lateralis and C. dalmaticus was in their tree choices of different decay level. Colonies of Camponotus dalmaticus were detected in rotten wet tree logs and in dead rotten tree roots, these were dispersing in surrounding ground. In total 60% of the Camponotus dalmaticus colonies existed in rotten logs or in underground dead tree roots. In Dinaric regions with sub Mediterranean climate Camponotus lateralis group species show similar distribution pattern to the Mediterranean populations. And in Dinaric habitats with continental climate influence, only Camponotus piceus was present. Camponotus lateralis and C. dalmaticus inhabit woodland and scrub land habitats with very little direct sun radiation. In hot Mediterranean climate Camponotus lateralis 81 Diversity and Nesting Preferences of Camponotus lateralis and C. dalmaticus are building colonies more often in wood, then in soil. By using wood for colonies constructions investigated species were probably compensating lower temperatures in shaded woodland habitats. Species using rocks, stumps, or logs as nest sites are vulnerable to low humidity and high temperature than those nesting in the soil (Gösswald, 1938). Chen et al., (2002) found that shaded, woodland, habitats of Camponotus vicinus have lower temperatures and greater humidity in comparison to open habitats. In our opinion Camponotus lateralis in woodland habitats build colonies more often in wood and over ground to compensate higher humidity in shaded habitats. Open grassland Mediterranean habitats are characterized by high ground temperatures due higher insulations and also by higher temperature variation (Cros et al., 1997). In the Mediterranean and Dinaric regions, we found Camponotus piceus colonies in scrub land and grassland habitats. Colonies of Camponotus piceus were built uniformly without ant hill and with one inconspicuous opening in ground. In the Mediterranean habitats only one colony of Camponotus piceus was built under stone and in the Dinaric region six colonies were found under stones. The colonies in ground provide protection from high day temperatures in exothermic grasslands that are inhabited by Camponotus piceus. In soil, below ground, humidity and temperature have a low degree of variability (Hölldobler and Wilson, 1990). In the Dinaric habitats colonies of Camponotus piceus were built on grounds with higher inclination, southern expositions and often under stone. It is possible that Camponotus piceus also use stones for faster colony heating, like some other cold adapted ant species (Hölldobler and Wilson, 1990). Differences in colony construction sites betweenCamponotus piceus and C. piceus sp. 2 were analysed. Data indicate that Camponotus piceus sp. 2 build colonies in ground without anthill. Colonies of Camponotus piceus sp. 2 were on habitats with larger inclination and with 40% to 80% stone coverage. Camponotus piceus sp. 2 colonies and workers were more often collected in the Mediterranean regions on higher elevations over 300 meters and in the Dinaric regions over 400 meters. Findings of Camponotus piceus sp. 2 colony on Mt. Velez was finding ofCamponotus piceus with highest elevation over 800 meters. In the context of altitudinal and thermal preferences within Camponotus lateralis group we find thatC. piceus have the widest ecological valence. Literature data indicate that Camponotus piceus also has greater northern distribution compared to Camponotus lateralis and C. dalmaticus (Radchenko, 1997). The results of our studies indicate ecological niche partitioning in the dimension of nest construction between three closely related Camponotus lateralis group species. Nest constructing differences between analysed species show spatial allocation. In the Mediterranean regions the competition between queens of Camponotus lateralis group probably maintain detected differences in the place of colony construction. In the Dinaric regions the main factor of absence of Camponotus lateralis and C. dalmaticus was temperature. 82 VESNIC, A., SKRIJELJ, R., TROZIC-BOROVAC, S., TOMANOVIC, Z. REFERENCES

Agosti, D., Collingwood, C. A., 1987, A provisional list of the Balkan ants (Hym. Formicidae) with a key to the worker caste. II. Key to the worker caste, including the European species without the Iberian. Mitteilungen der Schweizerischen Entomologischen Gesellschaft, 60: 261-293. Borowiec, L., Salata, S., 2013, Ants of Greece-additions and corrections (Hymenoptera: Formicidae). Genus, 24(3-4): 335-401. Bračko, G., Gomboc, M., Lupše, B., Marić, R., Pristovšek, U., 2014, New faunistic data on ants (Hymenoptera: Formicidae) of the southern part of Montenegro. Natura Sloveniae, 16(1): 41-51. Brian, M. V., Brian, A. D., 1951, Insolation and ant population in the west of Scotland. Transactions of the Royal Entomological Society of London, 102(6): 303-330. Chen, Y., Hansen, L. D., Brown, J. J., 2002, Nesting sites of the carpenter ant, Camponotus vicinus (Mayr) (Hymenoptera: Formicidae) in northern Idaho. Environmental Entomology, 31(6): 1037-1042. Colwell, R. K., Brehm, G., Cardelús, C. L., Gilman, A. C., Longino, J. T., 2008, Global warming, elevational range shifts, and lowland biotic attrition in the wet tropics. Science, 322(5899): 258-261. Cros, S., Cerdá, X., Retana, J., 1997, Spatial and temporal variations in the activity patterns of Mediterranean ant communities. Ecoscience, 269-278. Gösswald, K., 1938, Über den Einfluß von verschiedener Temperatur und Luftfeuchtigkeit auf die Lebensäußerungen der Ameisen. Zeitschrift fur Wissenschaftliche Zoologie, 151(3): 337-381. Heinrich, B., 1993, The Hot-blooded Insects: Strategies and Mechanisms of Thermoregulation. Harvard University Press, 600. Hölldobler, B., Wilson, E. O., 1990, The Ants. Harvard University Press, 732. Kaudewitz, F., 1955, Zum Gastverhaltnis zwischen Crematogaster scutellaris Ol. mit Camponotus lateralis bicolor Ol. Biologisches Zentralblatt, 74: 69-87. Lach, L., Parr, C. L., Abott, K. L., 2010, Ant Ecology. Oxford University Press, 402. Müller, G., 1923, Le formiche della Venezia Guilia e della Dalmazia. Bolletino della Societa Adriatica di Scienze Naturali, 28: 11-180. Pontin, A. J., 1960, Field experiments on colony foundation by Lasius niger and L. flavus (Hym., Formicidae). Insectes Sociaux, 7: 227-230. Porter, S. D., 1988, Impact of temperature on colony growth and developmental rates of the ant, Solenopsis invicta. Journal of Insect Physiology, 34(12): 1127-1133. Radchenko, A. G., 1997, A review of ants of the subgenus Myrmentoma, genus Camponotus (Hymenoptera, Formicidae), from Asian Palaearctic. Entomological Review, 77(8): 1050-1059. Seifert, B., 2007, Die Ameisen Mittel-und Nordeuropas. Tauer: Lutra Verlags-und Vertriebsgesellschaft, 368. Zimmermann, S., 1934, Beitrag zur Kenntnis der Ameisenfauna Süddalmatiens. Verhandlungen der Zoologisch-Botanischen Gesellschaft in Wien, 84: 5-65.

Received: February 19, 2016 Accepted: January 19, 2017 J. Entomol. Res. Soc., 19(2): 83-93, 2017 ISSN:1302-0250

Effects of Indole-3-Acetic Acid on Hemocytes of Achoria grisella Fabr. (Lepidoptera: Pyralidae)

Dilek ÇELİK Rabia ÖZBEK Fevzi UÇKAN*

Department of Biology, Faculty of Science and L i t e r a t u r e , K o c a e l i U n i v e r s i t y , 4 1 3 8 0 , K o c a e l i , T U R K E Y e-mails: [email protected], [email protected], *[email protected]

ABSTRACT In order to provide a more complete understanding of physiological impacts of IAA on insects, we investigated the cytotoxic effects of variable doses of IAA on hemocytes of Achoria grisella Fabr. (Lepidoptera: Pyralidae) larvae. The results revealed that addition of IAA (2 to 1,000 ppm) in diet of A. grisella larvae resulted in an increase in the total hemocyte counts at all doses tested. The percentage of plasmatocytes decreased but granulocytes increased at 2, 5, 100, 200 and 1,000 ppm. Nevertheless, the application of IAA did not alter mitotic indices, the percentage of spherulocytes, prohemocytes and oenocytoids. The percentage of living cells decreased at all treated doses compared to control interrelated with the elevated ratio of early apoptotic hemocytes at 5, 10, 50, 100 and 200 ppm. Significant reductions were observed at 2, 10, 50 and 100 ppm in the ratio of necrotic hemocytes, and at 10,100 and 200 ppm in the late apoptotic cells in IAA-treated A. grisella larvae. Our findings demonstrate that IAA exhibits detrimental effects on the hemocytes ofA. grisella larvae that are the main components of insect immunity. Key words: Achoria grisella, apoptosis, cytotoxic, hemocyte, indole-3-acetic acid, mitosis.

INTRODUCTION In the last fifty years, the world population multiplied more rapidly than even before. At the same rate, the requirement of agricultural production has increased and the application of plant growth regulators (PGRs), which promote agricultural production and prevent damage of pests, has become widespread (McDonald et al., 1988; Silva et al., 2003; Paulson et al., 2005). Auxins are a class of PGRs which are involved in many developmental processes like cell enlargement, cell division, vascular tissue differentiation and root initiation (Davies, 1995). Indole-3-acetic acid (IAA) isone of the most important natural auxins (Davies, 1995) which affects plant growth and development. However, it has also adverse impacts on non-target organisms such as insects by altering developmental time, longevity, reproductive potential and hemolymph metabolites (Rup et al., 2000, 2002; Kaur and Rup, 2003; Uçkan et al., 2011; Uçkan et al., 2014; Uçkan et al., 2015). The known harmful effects of insecticides have led to search for ecologically innocent compounds, which can repress pest insects. Authors have suggested that PGRs can be used instead of pesticides in Integrated Pest Management (IPM) 84 ÇELİK, D., ÖZBEK, R., UÇKAN, F. programs (Posnava, 1974; Kaur and Rup, 2002). However, the effects of these agents on different organisms still need to be clarified. The toxicological studies on the effects of PGRs including IAA have been conducted on different animals. De Melo et al., (1997, 2004) have suggested that IAA is cytotoxic for leukocytes by inducing necrosis and apoptosis. The adverse effects of PGRs on insects’ physiology have also been reported by using Bactrocera cucurbitae (Kaur and Rup, 2002), Spilarctia obliqua (Gupta et al., 2009), Galleria mellonella (Altuntaş et al., 2012; Er and Keskin, 2015), Apanteles galleria (Uçkan et al., 2014). The innate immune system of insects involves cellular and humoral defence (Strand, 2008b). Cellular defence system includes phagocytosis, encapsulation, nodulation and clotting that are directly mediated by immune cells called hemocytes (Lackie, 1988; Strand and Pech, 1995; Irving et al., 2005; Strand, 2008b). Hemocytes also play a role in humoral defence by producing soluble effector molecules (Imler and Bulet, 2005; Kanost and Gorman, 2008; Strand, 2008b). Another immune defence in insects is apoptosis, which occurs generally against viral infections (Clem, 2005) and can be also induced by several environmentally stimuli such as UV-irradiation or chemicals (Sun et al., 1999, Kim et al., 2001). Furthermore, cell death caused by toxic agents has a different morphology and is called necrosis (Wyllie, 1981). Because hemocytes are so sensitive against environmental impacts, the total count of hemocytes and apoptotic indices can be used as indicators for detecting cytotoxic effects of chemicals such as PGRs also stated by Altuntaş et al., (2012). The smaller wax moth, Achoria grisella Fabr. (Lepidoptera: Pyralidae) is ubiquitous pest of honey bee colonies. Only A. grisella at its larval stage feeds on honey combs and causes damage in apiculture (Chariere and Imdorf, 1997; Ellis et al., 2013). A. grisella larvae can also be used as model organisms model organisms in toxicological and immunological studies depending on its properties such as short life cycle, large hemolymph amount, easy and relatively cheap rearing conditions. Therefore, A. grisella larvae were selected as a model organisms to investigate the cytotoxic impacts of IAA. To our knowledge this is the first report that demonstrates total and differential hemocyte counts, and also mitotic and apoptotic indices of hemocytes in A. grisella larvae with and without IAA application. Our study will provide a more complete understanding of effects of IAA on insect hemocyte physiology and chemically- induced apoptosis.

MATERIALS AND METHODS

Insect rearing Stock culture of the A. grisella was reared with the temperature of 25± 2ºC, 60± 5% RH and 12:12 (L:D) photoperiod in our laboratory. The stock and successive cultures of A. grisella were established and maintained according to Uçkan and Gülel (2000) and Uçkan et al., (2011). 85 Effects of Indole-3-Acetic Acid on Hemocytes of Achoria grisella Bioassays Newly hatched A. grisella larvae were reared on IAA-treated artificial diet. Various doses (2, 5, 10, 50, 100, 200, 500 and 1,000 ppm) of IAA (Merck 10 g, Darmstadt, Germany) were added to distilled water and homogenized with artificial diet (Sak et al., 2006). The last instar larvae of A. grisella (45-50 mg) were used in all experiments.

Total and differential hemocyte counts In order to detect the impacts of various doses of IAA (2- 1,000 ppm) on the total hemocyte count (THC), last instars were washed in 70% ethyl alcohol and distilled water, and then air dried. After sterilization hemolymph was collected in anticoagulant buffer as described by Eret al., (2010). Ten microliters of these hemolymph suspension was used to count the total hemocyte number with a Neubauer Hemocytometer. To investigate the effects of various doses of IAA on differential hemocyte count (DHC), 5 µl of pure hemolymph was spread directly on sterile microscope slides and prepared for analysis according to Er et al., (2010). THC and DHC were assayed under an Olymphus BX51 (Olymphus Corp., Tokyo, Japan). All experiments were replicated five times with 3 larvae in each. Four hundred cells per larva were analysed for DHC.

Analysis of cell viability and mitotic indices The rate of dead and alive hemocytes, and mitotic indices of IAA-treated and untreated A. grisella larvae were investigated using acridine orange/ethidium bromide (Sigma Chemical Co.) double staining according to Er et al., (2010) and Altuntaş et al., (2012). Five microliters of hemolymph per larva and 10 microliters of dye cocktail were spread on slides, respectively and instantly examined under an Olymphus BX51 (Er et al., 2010, Altuntaş et al., 2012). Using double staining method, cell stages were identified as living cells, early apoptosis, late apoptosis and necrosis according to Cendoroglo et al., (1999) and (Kosmider et al., 2004). Three larvae were examined for each experimental group and replicated 5 times. Three hundred cells per larva were investigated for apoptotic, and mitotic indices.

Statistical analysis IAA-releated differences in the means were analysed using One-way Analysis of Variance (ANOVA) and comparison was done by Tukey’s Honestly Significant Difference (HSD) test when variances were homogenous; otherwise Tamhane T2 test. Percentage data were normalized by arcsine transformation prior to analysis. All data were analysed by the SPSS software (SPSS 18.0 for windows). Differences were considered statistically significant when P < 0.05.

RESULTS Addition of IAA (2- 1,000 ppm) to diet resulted in an increase in the total hemocyte number of A. grisella at all doses but the differences were significant at only 2 and 100 ppm when compared to the control (F=5.404; df=8, 126; P=0.000) (Table 1). The mean percentage of granulocyte of IAA- treated A. grisella increased at 2, 5, 100, 86 ÇELİK, D., ÖZBEK, R., UÇKAN, F. 200, and 1,000 ppm, but changes were not significant (F=1.758; df=8, 126; P= 0.092) (Table 2). On the other side, the percentage of plasmatocyte significantly decreased at 5 ppm when compared to the control, 10, 50, and 500 ppm (F= 2.240; df=8, 126; P= 0.029) (Table 2). In contrast, the application of IAA did not alter the percentage of spherulocytes (F=1.010; df=8, 126; P= 0.432), prohemocytes (F= 1.626; df=8, 126; P= 0.124), oenocytoids (F= 0.921; df=8, 126; P= 0.502) (Table 2) and also mitotic indices (F= 0.528; df=8, 126; P=0.833) (Table 1) significantly when compared to control. The mean percentage of living hemocytes of IAA treated A. grisella larvae declined at all tested doses, but the changes were not significant compared to the control and other doses (F=0.775; df=8, 126; P= 0.625) (Table 3). The mean early apoptotic hemocytes of IAA treated A. grisella significantly increased at 5, 10, 50, 100, and 200 ppm compared to control (F= 6.629; df=8, 126; P=0.000) (Table 3). However, IAA addition in A. grisella diet caused a decrease in the percentage of late apoptosis at 2, 10, 50, and 100 ppm (F= 3.599; df=8, 126; P=0.001) (Table 3) and necrosis at 5, 10, 50, and 100 ppm (F= 2.721; df=8, 126; P= 0.008) (Table 3). Table 1. IAA-related changes in total hemocyte counts and mitotic indices of A. grisella larvae. IAA Total hemocyte counta Mitotic indices a (x106 cell/ml) (Mean± SE)b (cells/300) (% ± SE)b (ppm)

0 6.38±0.38a 0.88±0.13a

2 9.55±0.56c 0.72±0.14a

5 6.91±0.42ab 0.92±0.20a

10 6.72±0.32ab 0.95±0.16a

50 7.19±0.31ab 0.75±0.15a

100 8.38±0.44bc 0.97±0.21a

200 8.08±0.45abc 0.80±0.18a

500 7.47±0.44ab 0.88±0.16a

1,000 7.26±0.39ab 0.78±0.16a aThe mean of 15 last instars per treatment. bColumns followed by the same letter (a-c) are not significantly different (P>0.05; Tukey’s HSD) The mean percentage of living hemocytes of IAA treated A. grisella larvae declined at all tested doses, but the changes were not significant compared to the control and other doses (F=0.775; df=8, 126; P= 0.625) (Table 3). The mean early apoptotic hemocytes of IAA treated A. grisella significantly increased at 5, 10, 50, 100, and 200 ppm compared to the control (F= 6.629; df=8, 126; P=0.000) (Table 3). However, IAA addition in A. grisella diet caused a decrease in the percentage of late apoptosis at 2, 10, 50, and 100 ppm (F= 3.599; df=8, 126; P=0.001) (Table 3) and necrosis at 5, 10, 50, and 100 ppm (F= 2.721; df=8, 126; P= 0.008) (Table 3). 87 Effects of Indole-3-Acetic Acid on Hemocytes of Achoria grisella

Table 2. IAA-related changes in percentage of differential hemocyte counts A. grisella larvae.

Differential hemocyte count (cells/400) (% ± SE)a

IAA (ppm) Hemocyte typeb

PLs GRs SPs PRs OEs

0 75.17 ± 1.67ac 20.22±1.95a 0.60±0.18a 2.88±0.48a 1.13±0.20a

2 71.35 ± 1.98abc 24.42±1.87a 0.78±0.16a 2.38±0.33a 1.07±0.28a

5 69.20 ± 1.87b 26.82±1.86a 0.90±0.22a 2.18±0.52a 0.90±0.22a

10 76.32 ± 1.34a 20.00±1.47a 0.52±0.08a 1.82±0.38a 1.35±0.19a

50 75.98 ± 1.76a 20.47±1.72a 0.45±0.13a 2.12±0.34a 0.98±0.24a

100 70.92 ± 1.83bc 25.07±1.87a 0.53±0.15a 2.45±0.45a 1.03±0.17a

200 71.22 ± 2.28ab 23.85±2.39a 0.68±0.19a 3.12±0.49a 1.13±0.20a

500 76.05 ± 1.90a 21.30±1.82a 0.48±0.13a 1.30±0.24a 0.87±0.16a

1,000 71.88 ± 1.73ab 24.23±1.65a 0.68±0.23a 2.52±0.40a 0.68±0.15a aThe mean of 15 last instars per treatment. bColumns followed by the same letter (a-c) are not significantly different (P>0.05; Tukey’s HSD; PLs, Plasmatocytes; GRs, Granulocytes; SPs, Spherulocytes; PRs, Prohemocytes; OEs,Oenocytoids)

Table 3. IAA-related changes in percentage of viable, early apoptosis, late apoptosis, necrosis of A. grisella larval hemocytes.

Apoptotic indicesa (cells/300) (%± SE)b

IAA (ppm) Viable Early Apoptosis Late Apoptosis Necrosis

0 55.22±4.19a 34.80±3.86a 7.00±1.44a 2.98±0.77a

2 53.87±3.46a 37.67±3.13a 6.91±1.54a 1.56±0.40bc

5 47.84±3.94a 45.87±3.92b 4.09±1.51ab 2.20±0.64acd

10 49.96±3.61a 46.22±3.55 b 2.98±0.80 b 0.84±0.17b

50 45.04±4.48a 49.36±3.81 b 4.67±1.41ab 0.93±0.23bd

100 47.09±3.34a 48.02±3.17 b 3.49±0.56 b 1.40±0.29bd

200 49.47±3.45a 45.56±3.68 bc 3.18±0.55 b 1.80±0.34ab

500 50.40±3.53a 41.00±2.65a 5.96±1.31a 2.65±0.50ac

1,000 47.98±2.86a 43.44±2.33ac 6.84±1.62a 1.73±0.39ab aThe mean of 15 last instars per treatment. bColumns followed by the same letter (a-d) are not significantly different (P> 0.05; Necrosis: Tamhane test, Other data sets: Tukey’s HSD).

DISCUSSION We had previously determined the impacts of IAA on the developmental characteristics of Apanteles galleria, G. mellonella and Pimpla turionellae (Uçkan et 88 ÇELİK, D., ÖZBEK, R., UÇKAN, F. al., 2011; 2015). In order to characterise further effects of IAA on insect immunity, IAA induced alterations in THCs, DHCs, mitotic and apoptotic indices of A. grisella hemocytes were investigated. Five distinct classes of hemocytes have already been identified in different Lepidopteran species, such as Manduca sexta (Horohov and Dunn, 1982), Poekilocerus bufonius (Al-Robai et al., 2002), Bomby xmori (Ganie et al., 2015), G. mellonella (Altuntaş et al., 2012), Spodoptera litura (Sharma et al., 2003), Ostrinia furnacalis (Jian et al., 2003), and Spodoptera littoralis (Ghoneim et al., 2015). In the present study the former five main hemocyte types were also identified in the hemolymph of last instars of A. grisella: prohemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. All of the treated IAA doses caused an increase in THC of A. grisella. Several reports have suggested that stress, wounding or infection can cause an increase in the number of hemocytes in circulation (Ratcliffe et al.,

1985; Lackie, 1988; Strand 2008b). Similarly, Altuntaş et al., (2012) reported that, GA3 caused a rise in THC in G. mellonella larvae. George and Ambrose announced (2004) that exposing to an organophosphate led to an increase in total hemocyte count and granulocyte numbers but a decrease in prohemocyte and plasmatocyte numbers of Rhynocoris kumarii. Similarly, we found that treatment with IAA in diet of A. grisella caused a decrease in the percentage of plasmatocytes whilst an increase in that of granulocytes. None of the applied doses led to significant changes in the percentage of spherulocyte, prohemocyte, and oenocytoids when compared to the control. Likewise, a various botanical originated insecticides such as azadirachtin (Azambuja et al., 1991; Er et al., 2017) and extract of Artemisia annua (Zibaee and Bandani) also alter hemocyte number of insects (James and Xu, 2012). Several reports in Lepidopteran demonstrated that the origins of hemocyte population in circulation during larval stage are both hemocytes, which can proliferate and differentiate, and hematopoietic organs (Strand 2008b). Our results revealed insignificant fluctuation in mitotic indices of hemocytes of IAA-treated A. grisella. Furthermore, the mitotic index fluctuations were not synergistic with dose depended increases in THC. Similarly, Altuntaş et al.,

(2012) demonstrated that GA3 treatment in G. mellonella larvae caused an increase in mitotic indices, but there was no correlation between induced mitotic indices and THC. As a typical Lepidopteran insect, Bombyx mori has four hematopoietic organs, which produce hemocytes and continuously release into the hemolymph during larval stages (Strand, 2008a, 2008b). It seems likely that, the source of incremental THC of IAA-treated A. grisella may be hematopoietic organs instead of mitosis of hemocytes already in circulation. Some evidences have suggested that there is a bidirectional connection between the immune system and nervous system (CNS) in insects (Beckage, 2008). Moreover, CNS regulates directly or indirectly insect hormones, which affect immune responses (Niijhout, 1994; Adamo, 2006; Beckage, 2008). Ecdysteroids and juvenile hormones (JHs) regulate many developmental and physiological processes including immune responses (Gade et al., 1997; Rantala et al., 2003; Franssens et al., 2006; Flatt et al., 2008). Several reports have suggested that the endocrine organs regulate hemocyte populations and differentiation (Riziki, 89 Effects of Indole-3-Acetic Acid on Hemocytes of Achoria grisella 1957, 1962; Hoffman, 1970; Judy and Marks, 1971; PrasadaRaoet al., 1984; Ahmad and Khan, 1988). Previously, we have demonstrated that IAA treatment led to a delay in larval developmental time of G. mellonella and immature developmental time of parasitoid P. turionellae, which are regulated by ecdysteroids and JHs. The observations presented here and previously described have led us to conclude that IAA may alter hormone balance between ecdysteroids and JHs and cause alternations in THC and DHC in A. grisella. As also stated above, the environmentally and internally regulatory stimuli (Kim et al., 2001) such as DNA damage, growth factor deprivation, death receptor signaling, radiation, chemicals or injury may cause apoptosis or necrosis (Dragovich et al., 1998; Sun et al., 1999; Bezabeh et al., 2001; Opferman and Krosmeyer, 2003; Nikoletopoulou et al., 2013). Our results indicated that whereas early apoptotic cells increased at low doses of IAA, the percentage of late apoptosis and necrosis decreased when compared to untreated larvae. Furthermore, there was a significant reduction in the percentage of living cells at all treated doses. Apoptosis inductive effect of IAA was also observed by Frukawa et al., (2004) in different tissue cells of mice. Altuntaş et al., (2012) also reported that GA3 treatment on G. mellonella larvae induced apoptotic and necrotic cell death and reduced cell viability when compared to untreated larvae. Microbial infections or pollutants/toxins such as UV radiations, pesticides, and ozone trigger production of reactive oxygen species (ROS) (Kohen and Nyska, 2002; Poljsak et al,. 2013). When ROS is not sufficiently reduced by antioxidant enzymes (Turrens, 2003 ; James and Xu, 2012), they may affect adversely on the organism by reacting with macromolecules of biological importance (James and Xu, 2012). Increased oxidative stress causes death of cells either by necrosis or by apoptosis (Zamzami et al., 1995, 1996; Tan et al., 1998; Kannan and Jain, 2000; James and Xu, 2012). A wide variety of synthetic insecticides are known to supress the activity of antioxidant enzymes (James and Xu, 2012). Olivera et al., (2007) reported that, IAA treatment on rat liver caused decrease in the activity of catalase and glutathione S- transferase. Likely, Çelik and Tuluçe (2006) reported that, IAA and kinetin increased the production of lipid peroxidases, and inhibited antioxidant defence in various rat tissues. Therefore, IAA treatment on A. grisella may cause a decrease in antioxidant enzyme activity and induce apoptosis. The increased interest to use eco-friendly products to promote agricultural production and to prevent damage of pests led us to investigate effects of ecologically innocent chemicals such as IAA in the model A. grisella larvae. Our data demonstrated that IAA affected THC and DHC, but has not effect on mitotic indices. In addition, IAA treatment caused a decrease in the percentage of living cells, late apoptosis and necrosis, but an increase in early apoptosis of A. grisella larval hemocytes. IAA showed similar effect with synthetic insecticides, which is harmful for target and non-target organism. Effects of IAA on other physiological parameters of insects such as hemolytic and phenoloxidase activity and antioxidant enzyme activity should also be explored before suggesting this plant derived compound as a good candidate for pest control in IPM programs. 90 ÇELİK, D., ÖZBEK, R., UÇKAN, F. REFERENCES

Adamo, S. A., 2006, Comparative psychoneuroimmunology: Evidence from the insects. Behavioral Cognitive Neuroscience Review, 5(3): 128-140. Ahmad, A., Khan, M. A., 1988, Effects of triol and makisterone A on the hemocytes of Hieroglymphus nigrorepletus Boliver (Orthoptera: Acrididae). Proceedings of the Indian Academy of Sciences (Animal Sciences), 97(3): 203-210. Al-Robai, A. A., Assgaf, A. I., Edrees, N. O., 2002, Study on types, total and differential haemocytes counts of Usherhopper, Poekilocerus bufonius Klug. Journal of King Saud University-Science, 14: 39-50. Altuntaş, H., Kılıç, A. Y.¸ Uçkan, F., Ergin, E., 2012, Effects of gibberellic acid on hemocytes of Galleria mellonella L. (Lepidoptera: Pyralidae). Environmental Entomology, 41(3): 688-696. Beckage, N. E., 2008, Insect Immunology, 1st edn. Academic press, San Diego, USA, 129-144. Bezabeh, T., Mowat, M. R. A., Jarolim, L., Greenberg, A. H., Smith, I. C. P., 2001, Detection of drug-induced apoptosis and necrosis in human cervical carcinoma cells using 1H NMR spectroscopy. Cell Death and Differentiation, 8(3): 219 -224. Cendoroglo, M., Jaber, B. L., Balakrishnan, V.S., Perianayagam, M., Kin,g A.J., Pereira, B. J. G., 1999. Neutrophil apoptosis and dysfunction in uremia. Journal of the American Society of Nephrology, 10(1): 93-100. Chariere, J. D., Imdorf, A., 1997, Protection of Honeycombs from Wax Moth Damage. Swiss bee research canter Federal Dairy Research Station Leibefeld CH-3003, Communication. Nr. 24, Liebefeld, Bern, Switzerland, 14pp. Clem, R. J., 2005, The role of apoptosis in defense against baculovirus infection in insects. Current Topics in Microbiology and Immunology, 289: 113-129. Çelik, I., Tuluçe, Y., 2006, Effects of indole acetic acid and kinetin on lipid peroxidation and antioxidant defense in various tissues of rats. Pesticide Biochemistry and Physiology, 84(1): 49-54. Davies, P. J., 1995, Plant Hormones, Physiology, Biochemistry, and Molecular Biology. 2nd edn. Kluwer Academic Publishers, Dordrecht, Netherlands, 1-35. de Azambuja, P., Garcia, E. S., Ratcliffe, N. A., WarthenJr, J. D., 1991, Immune-depression in Rhodnius prolixus induced by the growth inhibitor, Azadirachtin. Journal of Insect Physiology, 37(10): 771-777. de Melo, M. P., de Lima, T. M., Pithon-Curi, T. C., Curi, R., 2004, The mechanism of indole acetic acid cytotoxicity. Toxicology Letters, 148(1): 103-111. de Melo, M. P., Pithon-Curi, T. C., Curi, R., Di Mascio, P., Cilento, G., 1997, Peroxidase activity may play a role in the cytotoxic effect of indole acetic acid. Photochemistry and Photobiology, 65(2): 338-341. Dragovich, T., Rudin, C. M., Thompson, C. B., 1998, Signal transduction pathways that regulate cell survival and cell death. Oncogene, 17(25): 3207- 3213. Ellis J. D., Graham, J. R., Mortensen A.,2013, Standard methods for wax moth research. Journal of Apicultural Research, 52(1): 1-17. Er, A., Uçkan, F., Rivers, D. B., Ergin, E., Sak, O., 2010, Effects of parasitization and envenomation by the endoparasitic wasp Pimpla turionellae (Hymenoptera: Ichneumonidae) on hemocyte numbers, morphology, and viability of its host Galleria mellonella (Lepidoptera: Pyralidae). Annals of the Entomological Society of America, 103: 273-282. Er, A., Keskin, M., 2015, Influence of abscisic acid on the biology and hemocytes of the model insectGalleria mellonella (Lepidoptera: Pyralidae). Annals of the Entomological Society of America, 109(2): 244-251. Er, A., Taşkıran, D., Sak, O., 2017, Azadirachtin-induced effects on various life history traits and cellular immune reactions of Galleria mellonella (Lepidoptera: Pyralidae). Archives of Biological Sciences, 69(2): 335-344, DOI:10.2298/ABS160421108E. Flatt, T., Heyland, A., Rus, F., Porpiglia, E., Sherlock, C., Yamamoto, R., Garbuzov, A., Palli, S. R., Tatar, 91 Effects of Indole-3-Acetic Acid on Hemocytes of Achoria grisella

M., Silverman, N., 2008, Hormonal regulation of the humoral innate immune response in Drosophila melanogaster. The Journal of Experimental Biology, 211(16): 2712-2724. Franssens, V., Smagghe, G., Simoneta, G., Claeysa, I., Breugelmansa, B., De Loofa, A., Vanden B., J., 2006, 20-Hydroxyecdysone and juvenile hormone regulate the laminarin induced nodulation reaction in larvae of the flesh fly,Neobellieria bullata. Developmental and Comparative Immunology, 30(9): 735-740. Furukawa, S., Abe, M., Usuda, K., Ogawa, I., 2004, Indole-3-Acetic acid induces microencephaly in rat fetuses. Toxicologic Pathology, 32(6): 659-667. Gade, G., Hoffman, K. H., Spring, J. H., 1997, Hormonal regulation in insets: facts, gaps, andfuture directions. Physiological Reviews, 77(4): 963-1032. Ganie, N. A., Afifa, S. K., Baqual, M. F., Sharma, R. K., Dar, K. A., Bashir, M., 2015, Studies on the total and differential haemocyte count in some breeds of silkworm, Bombyx( mori L.). International journal of Advanced Biological Research, 5(1): 58-61. George, P. J. E., Ambrose, D. P., 2004, Impact of insecticides on the hemogram of Rhynocoris kumarii Ambrose and Livingstone (Hem., Reduviidae). Journal of Applied Entomology, 128(9-10): 600-604. Ghoneim, K., Tanani, M., Hamadah, K., Basiouny, A., Waheeb, H., 2015, Effects of Novaluron and Cyromazine, chitin synthesis inhibitors, on the larval hemogram of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae). International Journal of Advanced Research, 3(1): 554-576. Gupta, G., Yadav, S. R., Bhattacharya, A. K., 2009, Influence of synthetic plant growth substances on the survivorship and developmental parameters of Spilarctia obliqua Walker (Lepidoptera: Arctiidae). Journal of Pesticide Science, 82(1): 41-46. Hoffmann, J. A., 1970, Régulations endocrines de la production et de la différenciation des hémocytes chez un insecte Orthoptère: Locusta migratoria migratoroides. General and Comparative Endocrinology, 15(2): 198-219. Horohov, D. W., Dunn, P. E., 1982, Changes in the circulating haemocyte population of Manduca sexta larvae following injection of bacteria. Journal of Invertebrate Pathology, 40(3): 327-339. Imler, J. L., Bulet, P., 2005, Antimicrobial peptides in Drosophila, structures, activities and gene regulation. Chemical immunology and allergy, 86;1-21. Irving, P., Ubeda, J., Doucet, D., Troxler, L., Lagueux, M., Zachary, D., Hoffmann, J., Hetru, C., Meister, M., 2005, New insights into Drosophila larval haemocyte functions through genomewide analysis. Cellular Microbiology, 7(3): 335-350. James, R. R., Xub, J., 2012, Mechanisms by which pesticides affect insect immunity. Journal of Invertebrate Pathology, 109(2): 175-182. Jian, H., Xiang, X. Z., Wen, J. F., 2003, Passive evasion of encapsulation in Macrocentrus cingulum Brischke Hymenoptera: Braconidae), Apolyembryonic parasitoid of Ostrinia frunacalis Guenée (Lepiodoptera: Pyralidae). Journal of Insect Physiology, 49(4): 367-375. Judy, K. J., Marks, E. P., 1971, Effects of ecdysterone in vitro on hindgut and hemocytes of Manduca sexta (Laepidopteraa). General and Comparative Endocrinology, 17(2): 351-359. Kannan, K., Jain, S. K., 2000, Oxidative stress and apoptosis. Pathophysiology 7(3): 153-163. Kanost, M. R., Gorman, M. J., 2008, Phenoloxidases in insect immunity. In: Beckage, N. E. (Eds.). Insect Immunology, 1st ed. Academic press, San Diego, USA, 69-96. Kaur, R., Rup, P. J., 2002, Evaluation of regulatory influence of four plant growth regulators on the reproductive potential and longevity of melon fruit fly Bactrocera cucurbitae. Phytoparasitica, 30(3): 224-230. Kaur, R., Rup, P. J., 2003, Influence of some plant growth regulators (PGR) on biochemical profile in the larvae of melon fruit flyBactrocera cucurbitae (Coquillett) (Diptera: Trypetidae). Entomon Trivandrum, 28(2): 89-95. Kim, E. J., Rhee, W. J., Park, T. H., 2001, Isolation and characterization of an apoptosis-inhibiting component from the hemolymph of Bombyx mori. Biochemical and Biophysical Research Communications, 285(2): 224-228. 92 ÇELİK, D., ÖZBEK, R., UÇKAN, F.

Kohen, R., Nyska, A., 2002, Oxidation of biological systems: Oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicologic Pathology, 30(6): 620-650. Kosmider, B., Zyner, E., Osiecka, R., Ochocki, J., 2004, Induction of apoptosis and necrosis in A549 cells by the cis-Pt(II) complex of 3-aminoßavone in comparison with cis-DDP. Mutation Research, 563(1): 61-70. Lackie, A. M., 1988, Haemocyte behaviour. Advances in Insect Physiology, 21: 85-178. McDonald, R. E., Greany, P. D., Shaw, P. E., Schroeder, W. J., Hatton, T. T., Wilson, C. W., 1988, Use of gibberellic acid for Caribbean fruit by (Anastrepha suspensa) control in grapefruit. In: Goren, R., Mendel, K., (Eds.) Proceedings of the Sixth International Citrus Congress. March 6-11, Rehovot, Israel: Balaban Publications, 1147-1152. Nijhout, H. F., 1994, Insect Hormones. 1th edn. Princeton University Press, Princeton, UK, 1-13. Nikoletopoulou, V., Markaki, M., Palikaras, K., Tavernarakis, N., 2013, Crosstalk between apoptosis, necrosis and autophagy, Biochimica et Biophysica Acta-Molecular Cell Research, 1833(12): 3448-3459. Oliveira, D. L., Pugine, S. M., Ferreira, M. S., Lins, G. P., Costa, E. J., De Melo, M. P., 2007, Influence of indole acetic acid on antioxidant levels and enzyme activities of glucose metabolism in rat liver. Cell Biochemistry and Function, 25(2): 195-201. Opferman, J. T., Korsmeyer, S. J., 2003, Apoptosis in the development and maintenance of the immune system. Nature Immunology, 4(5): 410-415. Paulson, G. S., Hull, L. A., Biddinger, D. J., 2005, Effect of a plant growth regulator prohexadione-calcium on insect pests of apple and pear. Journal of Economic Entomology, 98(2): 423-431. Poljsak, B., Šuput, D., Milisav, I., 2013, Achieving the balance between ROS and antioxidants: When to use the synthetic antioxidants. Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity, Article ID 956792, 1-11. Posnava, A. N., 1974, Plant growth regulators and their use in applied entomology. Trudy Vsesoyuznogo Nauchnoissledovateľskogo institua Zashchity Rastenii, 40: 135-145. Prasada Rao, C. G., Ray, A., Ramamurty, P. S., 1984, Effects of ligation and ecdysone on total hemocyte count in the tobacco caterpillar Spodoptera litura (Noctuidae: Lepidoptera). Canadian Journal of Zoology, 62(8): 1461-1463. Rantala, M. J., Vainikka, A., Kortet, R., 2003, The role of juvenile hormone in immune function and pheromone production trade-offs: a test of the immunocompetence handicap principle. Proceedings of the Royal Society B: Biological Sciences, 270(1530): 2257-2261. Ratcliffe, N. A., Rowley, A. F., Fitzgerald, S. W., Rhodes, C. P., 1985, Invertebrate immunity basic concepts and recent advances. International Review of Cytology, 97: 183-350. Riziki, M. T., 1957, Alterations in the hemocyte population of Drosophila melanogaster. Journal of Morphology, 100(3): 437-458. Riziki, M. T., 1962, Experimental analysis of hemocyte morphology in insects. American Zoologist, 2: 247-256. Rup, P. J., Sohal, S. K., Kaur, G., Dhillon, M., 2002, The influence of allelochemicals and plant growth regulators on emergence and development of mustard aphid, Lipaphis erysimi (Kalt.). Allelopathy Journal, 10(1): 53-58. Rup, P. J., Sohal, S. K., Sohi, R., Kaur, G., Sandhu, N., Gurm, S. K., Dhingra, P., Wadhwa, S. K., 2000, Influence of PGRs on carbohydrate content inLipaphis erysimi (Kalt.). Indian Journal of Experimental Biology, 38: 1066-1068. Sak, O., Uçkan, F., Ergin, E., 2006, Effects of cypermethrin on total body weight, glycogen, protein, and lipid contents of Pimpla turionellae (L.) (Hymenoptera: Ichneumonidae). Belgian Journal of Zoology, 136(1): 53-58. Sharma, P. R., Sharma, O. P., Saxena, B. P., 2003, Effect of neem gold on hemocytes of the tobacco armyworm, Spodoptera littura (Fabricius) (Lepidoptera: Noctuidae). Current Science-Bangalore, 84(5): 690-694. Silva, M. T. B., Costa, E. C., Boss, A., 2003, Control of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) larvae with insect growth regulators. Ciência Rural 33(4): 601-605. 93 Effects of Indole-3-Acetic Acid on Hemocytes of Achoria grisella

Strand, M. R., Pech, L. L., 1995, Immunological basis for compatibility in parasitoid-host relationships. Annual Review of Entomology 40(1): 31-56. Strand, M. R., 2008a, The insect cellular immune response. Insect Science, 15(1): 1-14. Strand, M. R., 2008b, Insect hemocytes and their role in Immunity. In: Beckage, N. E. (Eds.). Insect Immunology. 1st ed. Academic press, San Diego, USA, 25-48. Sun, X-M., MacFarlane, M., Zhuang, J., Wolf, B. B., Green, D. R., Cohen, G. M., 1999, Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. The Journal of Biological Chemistry, 274(8): 5053-5060. Tan, S., Wood, M., Maher, P., 1998, Oxidative stress induces a form of programmed cell death with characteristics of both apoptosis and necrosis in neuronal cells. Journal of Neurochemistry, 71(1): 95-105. Turrens, J. F., 2003, Mitochondrial formation of reactive oxygen species. Journal of Physiology, 55, 335–344. Uçkan, F., Haftacı, İ., Ergin, E., 2011, Effects of indol-3-acetic acid on biological parameters ofthe larval endoparasitoid Apanteles galleriae (Hymenoptera: Braconidae). Annals of the Entomological Society of America, 104(1): 77-82. Uçkan, F., Soydabaş, H. K., Özbek, R., 2014, Effect of Indol-3-acetic acid on the biochemical parameters of Achoria grisella hemolymph and Apanteles galleriae larva. Pakistan Journal of Biotechnology, 11(2), 163-171. Uçkan, F., Gülel, A., 2000, Effects of host species on some biological characteristics ofApanteles galleriae Wilkinson (Hymenoptera; Braconidae). Turkish Journal of Zoology, 24(Ek): 105-114. Uçkan, F., Özbek, R., Ergin, E., 2015, Effects of indol-3-acetic acid on the biology of Galleria mellonella (Lepidoptera: Pyralidae) and its endoparasitoid Pimpla turionellae ( Hymenoptera: Ichneumonidae). Belgian Journal of Zoology, 145(1): 49-58. Wyllie, A. H., 1981, Cell death: a new classification separating apoptosis from necrosis. In: Bowen I. D., Lockshin R. A. (Eds.). Cell Death in Biology and Pathology, 1st edn. Chapman and Hall, London and New York, 9-34. Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsch, T., Susin, S. A., Petit, P. X., Mignotte, B., Kroemer, G., 1995, Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. The Journal of Experimental Medicine, 182(2): 367-377. Zamzami, N., Susin, S. A., Marchetti, P., Hirsch, T., Gomez, M. I,, Castedo, M., Kroemer, G., 1996, Mitochondrial control of nuclear apoptosis, The Journal of Experimental Medicine, 183(4): 1533-1544.

Received: April 14, 2016 Accepted: June 23, 2017

J. Entomol. Res. Soc., 19(2): 95-100, 2017 ISSN:1302-0250

Three New Minotetrastichus (Hymenoptera: Eulophidae) Species Records from Transcaucasia

George JAPOSHVILI1,2 Viktor KOSTJUKOV3

1 Institute of Entomology, Agricultural University of Georgia, Agmashenebeli Alley #240, Tbilisi, GEORGIA 2 Invertebrate Research Center, Agladze #26, Tbilisi-0119, GEORGIA 3 All-Russian Research Institute of Biological Plant Protection, Krasnodar 350039, RUSSIA e-mails: [email protected] , [email protected], [email protected]

ABSTRACT Up to this report, only Minotetrastichus platanellus (Mercet, 1922) had been recorded from Georgia and Transcaucasia. We now report Minotetrastichus frontalis (Nees, 1834), Minotetrastichus loxotoma (Graham, 1961) and Minotetrastichus treron Graham, 1987 as new to Georgia and Transcaucasia, bringing the number of species of Minotetrastichus Kostjukov, 1977 in Georgia up to four. Diagnostic characters for distinguishing this genus from other genera belonging to subfamily Tetrastichinae are provided. Key words: Georgia, Transcaucasia, Tetrastichinae, Diagnosis, Minotetrastichus, Kostjukov.

INTRODUCTION The Lagodekhi Protected Areas, established in 1912 is one of the world’s best-preserved areas of mixed forest with a diversity of natural landscapes. It is located in Lagodekhi, in the extreme north-eastern part of the southern slopes of the Caucasus and extends across an altitudinal range of 590-3500 m. The Lagodekhi Protected Areas includes Lagodekhi Nature Reserve (19749 ha) and Managed Reserve (4702 ha) (APA 2016). Kostjukov (1977), based on the results of a comparative morphological study of the subfamily Tetrastichinae, founded 17 subgenera within Tetrastichus Haliday, including Minotetrastichus, with the type species Cirrospilus ecus Walker. Later Graham (1987) revised genera of the European Tetrastichinae; 28 genera were recognized as valid, including Minotetrastichus as a distinct genus. Minotetrastichus currently includes ten species: M. citriscapus (Kostjukov), M. curtiventris (Kostjukov) (Kostjukov and Kosheleva, 2006), M. frontalis (Nees), M. loxotoma (Graham), M. napomyzae (Domenichini), M. pallidocinctus (Gahan), M. platanellus (Mercet), M. prolongatus Graham, M. treron Graham and M. zeasmi Narendran (Noyes, 2016). Species of Minotetrastichus are parasites of leaf-mining Lepidoptera, Coleptera and Hymenoptera. 96 JAPOSHVILI, G., KOSTJUKOV, V. This study represents a taxonomic analysis of part of the insect material collected in the Lagodekhi protected areas using Malaise traps during the entire growing season of 2014.

MATERIAL AND METHODS Malaise traps in the Lagodekhi protected areas were set in the following vertical zonal sites: 1. Low zone of forest (450-750m), 2. Middle zone of forest (750-1250m), 3. High zone of forest (1250-1800m), 4. Subalpine forest (1800-2000m), 5. Subalpine fields and shrublands (2000-2500m), 6. Alpine zone (above 2500m). Malaise traps were obtained from BandN Entomological services (Fres and Fibms, 2017) Containers were filled with 80% ethanol and were checked and replaced every ten days. Collecting started on April 2, 2014 and lasted until November 7, 2014, although in alpine and subalpine areas collecting started later (subalpine 5 May 2014; alpine 23 May 2014) and completed earlier (6 October 2014) due to poor climatic conditions. Material was collected every 10 (±2) days then it was transferred to the laboratory and was critical point dried following Noyes (2016) and mounted on cards. Identification was undertaken by the second author, using modern keys and papers of original descriptions, and the collections of the Zoological Institute of the Russian Academy of Sciences (St. Petersburg) and All-Russian Research Institute of Biological Plant Protection (Krasnodar). All voucher specimens are deposited to the Entomological collection of the Agricultural University of Georgia, Tbilisi, Georgia. Information about synonymy and biology is given in Graham (1987) and the Universal Chalcidoidea Database (Noyes, 2016). Therefore we did not add these data to our paper, unless absent from Graham (1987) and Noyes (2016).

RESULTS

Diagnosis of genus (Table 1) Female Length: 1.0-1.9 mm Anterior margin of clypeus truncated or with a pair of low, rounded lobes. Propodeum with spiracles very small or minute, circular or nearly so, separated from hind edge of metanotum by their diameter or more. Antenna with funicle and clava each with three segments. Mid lobe of mesoscutum usually without a median line. The two longer setae of each cercus usually subequal in length, pale straight or slightly curved (in loxotoma and prolongatus longest seta is 1.6x times longer than next longest seta). Mesoscutum convex. Body with at least weak metallic tints, but in some mainly yellow forms of platanellus metallic tints are lacking. Male Length: 0.8-1.3 mm Differs from female as follows: Antennal scape with a vertical plaque that is situated in upper part; antennal flagellum with a 4 segmented funicle and a 3 segmented clava. 97 Three new Minotetrastichus species records from Transcaucasia Table 1. Characters for differentiate Minotetrastichus from other Tetrastichinae.

The species of genus Minotetrastichus The other species of subfamily Tetrastichinae Female and male 1. Length 0.8-1.9 mm Length 0.4-4.5 mm Submarginal vein of forewing with 1 dorsal seta Submarginal vein of forewing with 2-5 dorsal or propodeal spiracles moderate-sized, separated setae; propodeal spiracles very small subcircular from hind margin of metanotum by much less 2. or minute, separated by at least 1.5 times their than their major diameter; if very small or minute diameter from hind edge of metanotum; the setae (Chrysotetrastichus and Ootetrastichus) then one of each cercus are subequal in length seta of each cercus is much longer than the other 3. Anterior margin of clypeus truncate Anterior margin of clypeus bidentate Antenna with scape usually black, often with 4. Antenna with scape usually yellow or yellowish metallic tints Gall-forming insects (usually Cecidomyiidae), Leaf-mining Lepidoptera, Coleoptera and Hosts Acari (Eriophyidae) and Nematoda; free-living Hymenoptera larvae and eggs of insects

Species list of Minotetrastichus distributed in Lagodekhi reserve (Georgia)

Genus Minotetrastichus Kostjukov, 1977

Species Minotetrastichus frontalis (Nees, 1834)

Material examined: 2 ♂♂, Lagodekhi reserve, Mt Kudigora, 41˚51.351ꞌ N, 046˚17.564ꞌ E, 847 m asl (above sea level), malaise trap, 15-25.05.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 15-25.05.2014, G. Japoshvili and G. Kirkitadze; 2 ♀♀, Lagodekhi reserve, Mt Kudigora,41˚52.288 N, 046˚18.692 E, 1351 m asl, malaise trap, 4-14.06.2014, G. Japoshvili and G. Kirkitadze; 3 ♀♀, 2 ♂♂, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 15-25.06.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, 1 ♂, Lagodekhi reserve, Mt Kudigora, 41˚ 54.371’ N, 46˚ 20.004’ E, 2558m asl, malaise trap, 15-25.06.2014, Leg. G. Japoshvili and G. Kirkitadze; 4 ♀♀, 5 ♂♂, Lagodekhi reserve, Mt Kudigora,41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 25.06-5.07.2014, G. Japoshvili and G. Kirkitadze; 2 ♀♀, Lagodekhi reserve, Mt Kudigora, 41˚51.351ꞌ N, 046˚17.564ꞌ E, 847 m asl (above sea level), malaise trap, 5-15.07.2014, G. Japoshvili and G. Kirkitadze; 6 ♀♀, 6 ♂♂, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 5-15.07.2014, G. Japoshvili and G. Kirkitadze; 2 ♀♀, 1 ♂, Lagodekhi reserve, Mt Kudigora,41˚52.288 N, 046˚18.692 E, 1351 m asl, malaise trap, 15-25.07.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚ 51.149’ N, 46˚ 17.266’ E, 666 m asl (above sea level), malaise trap, 25.07-5.08.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, 1 ♂, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 25.07-5.08.2014, G. Japoshvili and G. Kirkitadze; 4 ♀♀, 2 ♂♂, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 5-15.08.2014, G. Japoshvili and G. Kirkitadze; 2 ♀♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 25.08-4.09.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, 3 ♂♂, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 5-14.09.2014, G. Japoshvili and G. Kirkitadze; 2 ♀♀, 3 ♂♂, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 15-27.09.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 27.09-6.10.2014, G. Japoshvili and G. Kirkitadze. Distribution: Austria, Bosnia Herzegovina, Bulgaria, Canada (Alberta, British Columbia), Canary Islands, Croatia, Czech Republic, Denmark, Finland, France, Germany, Hungary, Ireland (Northern and Republic of), Italy, Macedonia, Moldova, 98 JAPOSHVILI, G., KOSTJUKOV, V. Netherlands, Norway, Pakistan, Romania, Russia (Altai Kray, Krasnodar Kray, Stavropol Kray, Nizhniy Novgorod Oblast, Ul’yanovsk Oblast, Siberia), Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey, Ukraine, United Arab Emirates, UK (England), USA (Connecticut, Delaware, Maine, Massachusetts, Montana, Ohio, Wisconsin) (Noyes, 2016).

Minotetrastichus loxotoma (Graham, 1961)

Material examined: 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 5-15.07.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 5-15.08.2014, G. Japoshvili and G. Kirkitadze. Distribution: Hungary, Norway, Russia (Primor’ye Kray), Slovakia, Sweden, UK (England), Croatia (Noyes, 2016).

Minotetrastichus platanellus (Mercet, 1922)

Material examined: 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 15-25.06.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 25.06-5.07.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, 1 ♂, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 15-25.07.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, 1 ♂, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 25.07-5.08.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 25.08-4.09.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚ 51.149’ N, 46˚ 17.266’ E, 666 m asl (above sea level), malaise trap, 5-14.09.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, 3 ♂♂, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 15-27.09.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288ꞌ N, 046˚18.692ꞌ E, 1351 m asl, malaise trap, 27.09- 6.10.2014, G. Japoshvili and G. Kirkitadze. Distribution: Bulgaria, Czech Republic, France, Georgia, Germany, Greece, Hungary, Iran, Israel, Italy, Netherlands, Serbia, Slovakia, Turkey, Ukraine, UK (England) (Noyes, 2016), Russia (Kostjukov and Nagorniy, 2004; Kostjukov et al. 2004; Kovalenkov et al. 2004a; 2004b; Litvinenko, 2004; Nagornyi, 2004; 2006).

Minotetrastichus treron Graham, 1987

Material examined: 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288’ N, 46˚18.692’ E, 1351m asl, malaise trap, 15-25.06.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288’ N, 46˚18.692’ E, 1351m asl, malaise trap, 25.06-5.07.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288’ N, 46˚18.692’ E, 1351m asl, malaise trap, 5-15.07.2014, G. Japoshvili and G. Kirkitadze; 1 ♀, Lagodekhi reserve, Mt Kudigora, 41˚52.288’ N, 46˚18.692’ E, 1351m asl, malaise trap, 5-15.08.2014, G. Japoshvili and G. Kirkitadze. Distribution: Croatia, France, Sweden (Noyes, 2016).

DISCUSSION Four species of Minotetrastichus are found in Lagodekhi reserve (Georgia). Only M. platanellus was recorded for Georgia and Transcaucasia (Kostjukov, 1978) prior to 99 Three new Minotetrastichus species records from Transcaucasia our study. Minotetrastichus frontalis, M. loxotoma and M. treron are new records for Georgia and Transcaucasia. Before our study, M. treron was known only from Croatia, France and Sweden, and M. loxotoma from Hungary, Norway, Russia, Slovakia, Sweden, UK, Croatia (Noyes, 2016). Minotetrastichus frontalis and M. platanellus, in contrast, are widely known species from Europe (Noyes, 2016) and North Caucasus (Kostjukov, 1978; Kostjukov and Nagornyi, 2004; Khomchenko and Kostjukov, 2004; Kostjukov et al., 2004; Kostjukov and Kosheleva, 2006; Kovalenkov et al., 2004a; 2004b; Litvinenko, 2004; Nagornyi 2004; 2006). Malaise traps seem to be good means of collecting species of Minotetrastichus and other Tetrastichinae. About one hundred individuals of Minotetrastichus spp. were collected by us in our Malaise trap, about 30 individuals of genus Tamarixia, 2 individuals of Neotrichoporoides and 4 individuals for Hyperteles. We recommend them for future sampling of Minotetrastichus.

ACKNOWLEDGEMENTS We would like to thank to Prof. Robert Paxton, Martin Luter University, Halle, Germany for his kind help in improving the language and for his valuable comments. We also thank to Rustaveli National Science Foundation for their financial supports under ref: FR/221/7-110/13. Finally we express our gratitude to Mr Meri Salakaia and Mr Marine Batsankalashvili for their kind help in sorting material.

REFERENCES

APA, 2016, Lagodekhi protected areas. http://apa.gov.ge/en/ (08.01.2016). Fres, B. N., Fibms S. N., 2017, http://entomology.org.uk/index.htm (15.03.2017). Graham, MWR.deV., 1961. The genus Aprostocetus Westwood, sensu lato (Hymenoptera: Eulophidae); notes on the synonymy of European species. Entomologist’s Monthly Magazine, 97: 34-64. Graham, M. W. R. de V., 1987, A reclassification of the European Tetrastichinae (Hymenoptera: Eulophidae), with a revision of certain genera. Bulletin of the British Museum, 55(1): 1-392. Khomchenko, E. V., Kostjukov, V. V, 2004, Chalcids (Hymenotpera, Chalcidoidea) from Krasnodar Kray and Stavropol Kray. Information I: Tetracampidae, Eulophidae, Aphelinidae, Elasmidae, Trichogramati- dae. In: Nadykta, V., Ismailov, V., Levashova, G., Sugonjaev, E. (Eds.) Biological Plant Protection as the Basis for Agroecosystem Stabilisation. Krasnodar: 1: 71-78. Kostjukov, V. V., 1977, Comparative morphology of chalcids of the subfamily Tetrastichinae and the systematics of the genus Tetrastichus Haliday 1844 (Hymenoptera, Eulophidae). Entomologicheskoe Obozrenie, 56(1): 177-194. Kostjukov, V. V., 1978, Hymenoptera II. Chalcidoidea 13. Eulophidae (Tetrastichinae). In: Medvedev, G. S. (Ed.). Opredeliteli Nasekomykh Evropeyskoy Chasti SSR, 3: 430-467. Kostjukov, V. V., Khomchenko, E. V., Kosheleva, O. V., 2004, Chalcids (Hymenoptera, Eulophidae) from Stavropolsky Kray and Kubani. In: Nadykta, V., Ismailov, V., Levashova, G., Sugonjaev, E. (Eds.) Biologicheskaya Zashchita rasteniy - osnova stabilizatsii agroekosistem, Krasnodar: 2: 170-181. Kostjukov, V. V., Nagorniy, A. A., 2004, Comparative analysis of parasitoid complex of Phyllonorycter platani Stgr on Platanus sp. and Phyllonorycter blancardella Z. on Cydonia oblonga Mill. In: Nadykta, V., Ismailov, V., Levashova, G., Sugonjaev, E. (Eds.) Biologicheskaya Zashchita rasteniy - osnova stabilizatsii agrozkosistem, Krasnodar: 2: 189-193. 100 JAPOSHVILI, G., KOSTJUKOV, V.

Kostjukov, V. V., Kosheleva, O. V., 2006, Taxonomic status of the species of the Tetrastichus Haliday, 1844 (S.L.) described from the territory of USSR with Trjapitzinichus gen.n. In: Nadykta, V., Ismailov, V., Levashova, G., Sugonjaev, E. (Eds.) Biological Plant Protection as the Basis for Agroecosystem Stabilization. Krasnodar: 4: 103-112 Kovalenkov, V. G., Kostjukov, V. V., Tjuzina, N. M., Khomchenko, E. V., 2004a, Principles and results of improvement of plant protection fruit cultivars. In: Nadykt,a V., Ismaiolov, V., Levashova, G., Sugonjaev, E. (Eds.) Biological Plant Protection as the Basis for Agroecosystem Stabilization. Issue Krasnodar: 1: 184-189 Kovalenkov, V. G., Kostjukov, V. V., Tjuzina, N. M., Kazadaeva, S. V., 2004b, Experience in switching from man-caused chemical tactics in orchard protection to chemical tactics in orchard protection to integrated phytosanitary situation management. In: Nadykta, V., Ismailov, V., Levashova, G., Sugonjaev, E. (Eds.) Biological Plant Protection as the basis for Agroecosystem Stabilization. Krasnodar: 2: 275-289 Litvinenko, E., 2004, Species of insect and acari of soybean in central part of Krasnodar Kray. In Nadykta V., Ismailov V., Levashova G., Sugonjaev E. (Eds.) Biological Plant Protection as the basis for Agroecosystem Stabilization. Krasnodar: 1: 108-116 Nogornyi, A. A., 2004, New and known chalcid species (Hymenoptera: Chalcidodiea) for fauna of Russia - parasites of leafmining Phyllonoricter (Lepidoptera) in Krasnodar Kray. In: Nadykta, V., Ismailov, V., Levashova G., Sugonjaev E. (Eds.) Biological Plant Protection as the basis for Agroecosystem Stabilization. Krasnodar: 1: 94-96. Nagornyi, A. A., 2006, Types of areals of leaf-mining insect parasites in North-Western Caucasus. In: Nadykta, V., Ismailov, V., Levasheva, G., Sugonjaev, E. (Eds.) Biological Plant Protection as the basis for Agroecosystem Stabilization. Krasnodar: 4:112-116 Noyes, J. S., 2016, Universal Chalcidoidea Database. World Wide Web electronic publication. http://www. nhm.ac.uk/chalcidoids. (10.3.2016).

Received: May 06, 2016 Accepted: April 04, 2017 J. Entomol. Res. Soc., 19(2): 101-112, 2017 ISSN:1302-0250

Effects of Various Plant Extracts on the Development of the Potato Beetle under Laboratory and Field Conditions: A Combined Study

Ömer ERTÜRK1* Adnan SARIKAYA2

1*Department of Biology, Faculty of Arts and Sciences, Ordu University, 52750, Perşembe, Ordu, TURKEY, e-mail: [email protected] 2Department of Biology, Faculty of Arts and Sciences, Amasya University, 05100, İpekköy, Amasya, TURKEY, e-mail: [email protected]

ABSTRACT In this study, we examined the effects of ethanolic extracts obtained from the various parts ofLiquidambar orientalis, Buxus sempervirens, Alnus glutinosa, Artemisia absinthium, Aesculus hippocastanum and Rhus coriaria on the egg-laying behavior of the potato beetle, Leptinotarsa decemlineata, under laboratory conditions at 25±2°C and 60±5 % relative humidity, and tested the antifeedant and toxic effects of the two extracts leading to the smallest number of egg-laying, Liquidambar orientalis and Buxus sempervirens, with a field study. The field study showed that the average number of 6.2 larvae and 7.2 adults before treatment of the Liquidambar orientalis extract decrease to 1.2 larvae and 3.2 adults after treatment. With the use of the Buxus sempervirens extract, the 10.2 larvae and 8.4 adults that existed before the treatment were reduced to 2.6 larvae and 2.8 adults after treatment. As for the eggs laid in the plants, the control plants had 13 while Liqudambar orientalis decreased this number to 3 and Buxus sempervirens to 2. These results suggest that these two plant extracts can be used in the field as a potential alternative to chemical pesticides. Key words: Buxus sempervirens, botanical pesticides, Liquidambar orientalis, plant extracts, potato beetle, pest management.

INTRODUCTION Biotic and abiotic factors prevent the present agricultural areas from being used effectively. One of these biotic factors is that, pests are known to decrease the quality and productivity in forests and agricultural areas (Fenemore, 1984; Ecevit, 1988; Thacker, 2002; Isman, 2006; Matthews, 2006; Zehnder et al., 2007; Bianchi et al., 2013). Synthetic insecticides have been used for the last 50 years to control these pests. There are five main classes of synthetic insecticides; namely organochlorides, organophosphates and carbamates, pyrethroids, neonicotinoids and ryanoids (Ware, 1982; Dorow, 1993; Palmquist et al., 2011). The most commonly used one of these are the organochlorines. Developed countries have banned their use along with various other pesticides such as the synthetic DDT, therefore leading to a search for alternative methods in management of the pests (Franzen, 1993; Thacker, 2002; Scott et al., 102 ERTÜRK, Ö., SARIKAYA, A. 2003; Isman, 2006; Matthews, 2006; Zehnder et al., 2007). Management of pests in Turkey is undertaken largely by using chemicals, the use of which not only leads to chemical resistance in pests, but also cause the death of useful insects such as bees, or animals such as birds and fish. These chemicals may even reach humans via the food chain and eventually lead to a number of permanent or fatal diseases (Fenemore, 1984; Ecevit, 1988; FAO, 1992; Matthews, 2006; Handal et al., 2016). An alternative method of minimizing the harms of pests may be the use of poisonous and healthy plant extracts which can be found in nature (Alberto et al., 2010). These extracts contain anti-feedant substances and substances which regulate or inhibit beetle growth and development. They also contain substances inhibiting the effects of juvenile and the moulting hormones in beetles and/or substances imitating their functions (Whittaker and Feeny, 1971; Berenbaum, 1985). There are many plants which may be used as pesticides. Plants were used for the first time for pesticide activities in the mid-17th century (Smith and Secoy, 1975). These plants are seen today as a potential source of natural pesticides due to the various substances they contain. Plant pesticides are effective only on the pests themselves and they have the advantage of not harming non-targeted organisms or humans. Besides, they are easily degradable and environment-friendly (Berenbaum, 1985). As opposed to traditional pesticides which managed pests only with an active substance, plant pesticides contain extracts effective on both the behaviors and the physiologies of pests (Saxena, 1987). The main purpose of the agricultural studies is to increase the yield of product per hectare. The primary damage of Colorado potato beetle, Leptinotarsa decemlineata (Say, 1824) (Coleoptera: Chrysomalidae), is leaf feeding by larvae and adults, although young fruits can also be eaten when the hosts are eggplant or tomato. Leaf-feeding has the greatest effect on potato growth, if occurring within two weeks of peak flowering, whereas during the last few weeks before harvest or very early in the growth of the crop it has little effects on yield in Turkey. Up to now, chemical substances of the pyrethroid group, such as deltamethrin (Deltamethrin) have been utilized to control this pest (Ertürk and Uslu, 2007; Turkish Ministry of Agriculture and Rural Affairs, 2008). This study identified the effects of various plant extracts on this pest in a laboratory, and then examined the two most effective ones by spraying them on potatoes in a field to test the behavior of the potato beetle, its feeding, egg-laying, larvae and adult density.

MATERIAL AND METHODS

Selection and maintenance of the pest L. decemlineata larvae and adults were carefully collected between May and June 2002 in the areas used for growing cultures around Ordu province and its towns in Turkey. Without damaging the larvae and adults using a tool Spraying bottle, they were brought to the laboratory in plastic boxes allowed airflow. Throughout the study, 103 Effects of Various Plant Extracts on the Development of the Potato L. decemlineata larvae and adults fed on fresh potato leaves. The larvae and adults of the potato beetle destroyed the plant by affecting its leaves and young branches as well as by laying eggs. Therefore, these two features were selected for the evaluation of results (Ertürk and Uslu, 2007). Agria varieties were used as the potato variety in this study.

Selection of the plants In this study, we used extracts obtained from various parts of Buxus sempervirens, Aesculus hipopcostonum, Rhus coriaria, Alnus glutinusa and Artemisia absinthium. These plants collected from Black Sea Region of Turkey in 2003 in field.Liquidambar orientalis was bought from a herbalist. The identification of these plants was done according to Davis (1966-1988). The literature survey of plants used in this study has chosed. The literature survey of plants used in this study was conducted.

Preparation of plant extracts Fresh leaves, flowers and seeds were washed under running distilled water. The fresh stems and leaves of plants were dried at 45°C for 5-6 hours. The leaves from Lq. orientalis were cut into pieces and grinded into powdery form using pestle and mortar and shade dried. The extract of the leaves, flowers, and seeds were prepared according to the methods described (Holopainen et al., 1988; Yildirim et al., 2000; Mohan et al., 2009). The powders were stored in air tight plastic tube. Water and ethanolic extracts were prepared of sterile distilled water and ethanol mixing 250 gr of dry powder of plants and 1250 ml solvent, at room temperature in a round bottom flask with occasional shaking. The extracts were kept at 4°C for 5 days. Both water and ethanolic extracts were then filtered through a muslin cloth for coarse residue and they finally filtered through were filtered through 100 µm membrane filter, and then the solutions were dried with an evaporator. The crude extracts were stored at -20°C until they were used. Before their use, they were dissolved in 25% ethanol.

Bioassays

1. Laboratory study The varieties potatoes of agria, the seed potatoes, were used in this study. According to the Provincial Directorate of Ordu, it is the most suitable variety for this region. They were planted in plastic pots (approximately 35 cm wide and 45 cm deep). The soil used was chosen carefully and mixed with natural animal fertilizers. Then an equal number and weight of potatoes were planted in the pots which were kept under controlled laboratory conditions until they leaf out. All pots were exposed to normal weather conditions. All pots were kept at 25±2°C and 60±5 % relative humidity. The potted plants were covered with veils to avoid contamination from the outside extra insects. When the potato plants in the pots were grown enough, approximately four to five weeks after the potato plant height of 50 cm. 50 ml of each previously prepared plant extract (see, preparation of plant extracts) was transferred into plastic glass and 104 ERTÜRK, Ö., SARIKAYA, A. applied to the plants homogeneously. Two pots were formed for each plant extract. Following treatment, three pairs of L. decemlineata adults (six in total) were placed in each pot. In order to prevent beetle crossings between pots, each pot was tied by tulle cloth allowing airflow. When they arrive, plant height 50 cm, 2 male and 4 female was left on the plants healthy potato beetle and the beetles laid their eggs. After 7 days, plant extracts were applied for a second time in a similar to the first one. These pots were then controlled weekly and prepared experiment reports. All operations were performed under the same conditions all the same parent plant. Triple repetition.

2. Field study Field study was carried out in a quarter-acre potato growing area near the town named Kabadüz in Ordu province. The Kabadüz (40°59’01”N, 37°53’02”E) is located in the south of Ordu, at an altitude of about 600 meters with an annual average temperature of 4.3°C and average relative humidity of 74.7%. Soil before planting was organized as agricultural. The plants were watered individually with drip irrigation and fertilized according to the farmer planting. Potatoes were planted in groups of 25 and the experiment was made to coincide with the chemical spraying time. The field in farmland was divided into three plots separated by a barrier of 3-mm thick clear polyethylene film. The potatoes were planted in three blocks with 25 potato roots in each block. Each plot was further divided into three sections of 3 rows each, and each section served as a replicate for sampling. The potatoes were planted in the plot in mid-March 2006 in rows of 70 cm spacing, each with 25 plants spaced 40 cm apart. Experiment was designed with three replications and for each plant extract a total of 75 potato roots were used. These potatoes were grown until the experiment. Care was taken to ensure equal amount of plant extract application to foliage of potato plants. After determination of leaves on the ground and in the period following the first insect to be seen on a potato leaf, spray was applied in the field. Once every 7 days to monitor the population dynamics of L. decemlineata, larvae, adults and eggs were observed. A total of 4 samplings were conducted from 15 April 2006 to 15 May 2006. The control block and the blocks which were to receive the plant extract applications were labeled as Part 1, 2 and 3. The two extracts found to be most effective in the laboratory study (Lq. orientalis and B. sempervirens) were chosen to be used in the field study (Table 1). The plant extract to be tested in each block was prepared in the laboratory and transferred to the field in dark-colored bottles. Following the identification of the existence of L. decemlineata adults, larvae and eggs on the potato plants in the marked blocks, 250 g per liter plant extracts were applied homogeneously to each potato plant with a back pulverizator (about 10-15 liters capacity, a suitcase model). The control group was sprayed with the solvent (water and ethyl alcohol) in which plant extracts were dissolved. The solvent was diluted so as not to damage the plant. After a week, the experiment was repeated in the same way. Each block was controlled weekly and experiment reports were prepared. Samples of L. decemlineata, larvae, adults and eggs were randomly collected from each section in a paper towel to prevent moisture, 105 Effects of Various Plant Extracts on the Development of the Potato placed in plastic bags, and returned to the laboratory. The effectiveness of two extracts under field study was calculated with the Handerson-Tilton formula (Lamparski and Wawrzyniak, 2005).

Statistical analyses The statistical analyses of the data were performed by using the SPSS for Windows (version 12.0) package program. In order to determine the effects of the two plant extracts before and after treatment on the potato beetle’s larvae and adults, the significance of difference between groups was tested by one way variance analysis (one way ANOVA). When there was a difference between groups, the means was tested by post HOC tests (Tukey HSD). In the evaluation of data, if p-value is less than or equal 0.05, it was accepted as significant. Table 1. Parts used of plants and effect of its extracts on egg-laying behavior of potato beetle, Leptino- tarsa decemlineata, in laboratory conditions. Ecozone in Number of Situation of Plant used Family name Local name Part(s) used Turkey laying egg eggs Liquidambar Turkish sweetgum Muğla- 3 egg pocket Altingiaceae Rasin 45 orientalis (Turkish, Sığla ağacı) Fethiye clear yellowish Buxus Boxwood tree North 2 egg pocket Boxaceaea Seed and leaf 47 sempervirens (Turkish, Şimşir) Anatolia darker yellowish Alnus Common alder Northen Betulaceae Leaf 59 - glutinosa (Turkish, Adi kızılağaç) Anatolia Artemisia Wormwood The whole Asteraceae Flower and leaf 65 - absinthium (Turkish, Pelin otu) Anatolia Aerulus Horsechesnut The whole Sapindaceae Fruit and leaf 93 - hippocastanum (Turkish, At kestanesi) Anatolia Rhus Sumac West and Anacardiaceae Leaf 104 - coriaria (Turkish, Sumak) South Anatolia Control 13 egg pocket - - - - 9 (Water only) clear yellowish Control - - - - 0 - (Ethyl alcohol)

RESULTS Table 1 presents findings about the effects of the eggs ofL. decemlineata adults on the potato plants grown in laboratory pots. The table shows that, among the six different plant extracts used, the most successful extract against the egg-laying behavior of L. decemlineata was Lq. orientalis, and the least successful R. coriara (Table 1). The Lq. orientalis extract, which was not found to be toxic in beetles, acted as an effective anti-feedant thus preventing beetles from feeding and laying many eggs (Table 1). The beetles placed in the pot with this extract were not able to consume the majority of the plant’s leaves until harvesting, and not able to lay more eggs (Table 1). Those placed in the pot with B. sempervirens did not consume the majority of the plant’s leaves until harvesting and did not lay many eggs (Table 1). The A. hippocastanum extract was not able to prevent beetle eggs from hatching. This plant extract did not have an adequate anti-feedant effect on any life stages of the beetle. Thus, these beetles were well-fed and laid many eggs (Table 1). This extract was observed not to be very effective in the biological control against the potato beetle. The A. absinthium and A. glutinosa extracts had similar effects and led to the laying of 106 ERTÜRK, Ö., SARIKAYA, A. a similar number of eggs (Table 1). These extracts did not fully prevent adult beetles from destroying the leaves of the potato plant. Therefore, they were decided not to be useful in biological control. The R. coriaria extract was similarly found to be not very effective against the potato beetle (Table 1). Due to the excessive damage caused by the adult beetles in the control groups, the plants in these pots were left without leaves, which stopped beetles from laying their eggs. Lq. orientalis and B. sempervirens, the two plant extracts leading to the fewest number of L. decemlineata eggs in the laboratory, were chosen for field study. Table 2 and Figure 1 show the results of the field study. Before the treatment of the Lq. orientalis extract, the average number of larvae and adults was 6.2 and 7.2, respectively (Table 2), but after the treatment these numbers decreased to 1.2 and 3.2, respectively, (Table 1 and Figure 1). Also, a small number of eggs were identified in the 3 seedbeds. When compared to the eggs in the control block, the ones in this group were of a darker yellowish color and only 3 dead adult beetles were found in the whole area (Table 2). The appearance of potato seedbeds was respectable (Figure 2). In the potato blocks before the treatment of B. sempervirens, an average number of 10.2 larvae and 8.4 adults were seen. These values decreased to an average of 2.6 larvae and 2.8 adults after the treatment of the extract. A small number of eggs were identified in the two seedbeds. The appearance of the eggs resembled those in the block with Lq. orientalis (Table 1). In the count in this block, no adult beetles were seen. The potato seedbeds looked healthy (Figure 2). In the potato seedbeds sprayed with 25% ethanol (control group), the potato leaves were almost fully eaten, the number of larvae was dense, many eggs were laid, and the number of adult beetles too much that of the other two blocks. The potato seedbeds were damaged (Figure 2). While the numbers of larvae and adults were 10 and 9.6 respectively before the treatment of ethanol, the numbers were 10 and 7.4 after the treatment. Before and after the treatment, the difference in the numbers of both larvae and adults was statistically insignificant, (p < 0.05). Table 2. Effects on potato beetle, Leptinotarsa decemlineata of extracts obtained from Buxus sempervi- rens, and Liquidambar orientalis, in field.

Before treatment After treatment Plant Number of Larvae Number of Adults Number of Larvae Number of Adults (mean*±SEM)** (mean*±SEM)** (mean*±SEM)** (mean*±SEM)** Control 10.0±1.67 a 9.6±1.03 a 10.0±1.38 a 7.4±0.68 a

Liquidambar orientalis 6.2±1.02 b 7.2±0.58 b 1.2±0.49 b 3.2±0.37 b

Buxus sempervirens 10.2±1,56 a 8.4±0.51 a 2.6±0.75 b 2.8±0.58 b

*Each represents three repeats **The differences between the values in the same columm fallowed by the same letter are not statisti- cally significant, p>0,05.

Extraction efficiency Extract from Lq. orientalis was more effective than extract fromB. sempervirens. On the contrary, the effects of the extract from B. sempervirens at adults of the pest 107 Effects of Various Plant Extracts on the Development of the Potato were more than extract from Lq. orientalis. Efficiency of the extract fromLq. orientalis at larvae of the pest was 80.65 %, and was 42.34 % at adults of the pest. On the contrary, efficiency of the extract fromB. sempervirens at larvae of the pest was 74.51 %, and was 56.76 % at adults of the pest. In the potato seedbeds sprayed with 25% ethanol (control group), the potato leaves were almost fully eaten, while in the potato seedbeds sprayed with extract from B. sempervirens and Lq. orientalis the potato leaves were not fully eaten by potato beetle.

Fig. 1. General aspect of potato blocks after treatment in field: B. sempervirens (A), Liquidambar orientalis (B) Control (only ethanol solvent) (C).

A B 12 12

Before Treatment Before Treatment

10 After Treatment 10 After Treatment

8 8

6 6 Number of Adult of Number

Number of Larvae 4 4

2 2

0 0 Lq. orientalis B. sempervirens Control Lq. orientalis B. sempervirens Control

Fig. 2. Comparison results of before treatment and after treatment of extracts, number of larvae (A) and number of adult beetle (B).

DISCUSSION Natural substances found in plants are an efficient alternative for the chemicals used in the controls of phytophagous insects. These substances have been shown to suppress the growth potential of the pest as well as its feeding (Whittaker and Feeny, 1971; Berenbaum, 1985). Plant extracts generally realize these effects with the active substances they contain. The effects of these components on pests depend on many variables such as the solvent used or environmental factors such as the temperature, climate or humidity (Ertürk, 2002). As an added difficulty, determining the most active substance in these extracts is not always easy. The isolated extract 108 ERTÜRK, Ö., SARIKAYA, A. may not always have the desired effect (Van Beek and De Groot, 1986). The majority of these substances are thought to be anti-feedants. The chemicals in various plants which inhibit pest feeding are known as natural anti-feedants. Until today, triterpens, lactons, alkaloids, cucurbitacins, quinins and phenols have been used as anti-feedants in pest control (De Groot and Van Beek, 1987; Wieczorek, 1996). Nowadays, some plant pesticides are commonly used as food, spices or medical treatment in developed countries. For instance, in North America and European countries, plant pesticides such as Chrysanthemum cinerariifolium (Trevir.) Vis. pyrethrum, Derris spp. and Lonchocarpus spp. rotenone, Nicotiana tabacum L. nicotine, Ryania speciosa Vahl ryania and Azadirachta indica neem are used instead of chemical pesticides and are sold as agricultural products (Nawrot et al., 1986; Norris, 1986). The larvae and adults of the potato beetle damage the leaves of the plant with the eggs they lay on the leaves and young branches (Turkish Ministry of Agriculture and Rural Affairs, 2008). On GC–MS analysis of the Styrax liquidus from Lq. orientalis, overall, 31 compounds representing 99.8% of the total oil were identified where 1-8 Cineole, α-pinene, piperitone were identified as the major components by Gurbuz et al., (2013). Lee et al., (2004) reported that Eucalyptus nicholii and Metrosideros fulgens showed them to be rich in 1,8-cineole (82.19%, 77.50%, respectively) with lower amounts of other mono- and sesquiterpenoids. The oriental sweetgum from Lq. orientalis containing 1,8-cineole, α-pinene, piperitone concentrations showed the highly in toxicity in the presence of the larvae and adults of potato beetle. It is well known that variation in the composition of essential oils depends on genetics, type and age of leaf source, environment and oil analysis. Boland et al., (1991) also found that E. nicholii contained 1,8-cineole (84%), isovalic aldehyde and α-pinene, E. codonocarpa contained ρ-cymene (30%) and piperitone (39%), and E. blakelyi contained 1,8-cineole (55%), and limonene (11%). Under our methods of extraction and analysis essential oils from similar sources are similar in composition. Eucalyptus oil (E. globulus Labill.) and eucalyptol (1,8-cineole) are accepted as food flavourings in a number of countries (Ash and Ash, 1995). The extracts mentioned above suppress the appetite of the pests with their anti-feedant substances and strong smell. Not feeding enough, they therefore do not complete their development. The extracts also avoid the eggs from hatching, make them turn a darker color, inhibit the feeding of the larvae and adults, and reduce nutrient consumption by probably destroying the metabolism of the larvae. All of these prevent the potato beetle from eating the leaves of the plant. If the plant extract is effective against the pest, it is natural that the beetle will lay fewer eggs on the plants which receive a treatment of this extract. In our study, the pest was seen to lay the smallest number of eggs in the potato plants which received Lq. orientalis and B. sempervirens extracts, which protected the plants against the damage made by this pest (Table 1). Ertürk and Uslu (2007) have also identified these two extracts as the best anti-feedant for the potato beetle in a study conducted with 20 plant extracts in a laboratory. They were therefore used in the field study (Table 2). Ertürk and Uslu (2007) reported that these two extracts decreased the nutrient consumption of the potato beetle larvae and adults, and reduced 109 Effects of Various Plant Extracts on the Development of the Potato their body weight under laboratory conditions. They also showed that the toxicity level of these two extracts were high. However, they concluded that these extracts acted as antifeedants and repellents, more than their toxicity (Saxena, 1987; Rembold, 1994; Ertürk and Uslu, 2007). As is widely known, such components in plant drugs inhibit the effects of juvenile and the moulting hormones in insects and imitate their functions (Bowers et al., 1976). As the extracts studied here also contained similar substances, the number of the eggs and larvae in the experimental group was observed to be much smaller than the control group (Table 2 and Figure 2). The extracts had a toxic effect on eggs and thus decreased the number of larvae. Under field conditions, these two extracts also inhibited female pests’ oviposition (Table 1). Probably due to the aromatic components in two extracts, this may help the plant protect itself against massive damage from pests (Table 2). Mukherjee (2003) reported that the influence of seven plant allelochemicals on growth rate, nutritional physiology and mid-gut esterase activity was studied in fifth instar larvae of Spodoptera litura. Relative growth rates were highest in larvae fed on the control diet followed by diet treated with β-sitosterol (0.618± 0.012 mg d−1 mg−1) and cineole (0.618 ± 0.054 mg d−1 mg−1) (Abramson et al., 1973). Furthermore the activity of the leaves of B. sempervirens extracs is likely due to the high content of sitosterol, stigmasterol, cycloartenol, lupeol, germanicol and β-amyrin in the freestate. This is the first report of significant insecticidal activity by these extracts and compounds against the larvae and adults of potato beetle. Turkey that is known to have a very rich flora may use this potential natural resource in agriculture as well as in medical treatments, landscape architecture, constructions or as food and spices (Davis, 1966-1988). We are of the opinion that more studies of this nature will help a better understanding of the active substances in plant extracts and thus benefit agricultural areas. With similar studies, we may identify alternative substances to chemicals which contaminate our natural resources and threaten our future. In addition to reducing environmental pollution, the use of these cheaper natural pesticides would have an economic benefit as well. According to our field results, Lq. orientalis and B. sempervirens may be used as plant pesticides, due to the substances in their extracts, with the aim of inhibiting the population and feeding of L. decemlineata larvae (Table 2, Figure 2). Therefore, we offer to use together under field condition these two extracts according to pest’s development stages.

CONCLUSION Plants could be an alternative source to prevent damage to crops caused by the potato beetle because they constitute a potential source of bioactive chemicals and are generally free from harmful effects. Using of these botanical derivatives in potato beetle control instead of synthetic insecticides could reduce the cost and environmental pollution. Further studies on identification of active compounds, toxicity and field trials are needed to recommend the active fraction of these plant extracts for development of eco-friendly chemicals for control of insect vectors. These two plants extracts from 110 ERTÜRK, Ö., SARIKAYA, A. our study results, because of the secondary compounds they included, it showed toxic and lethal effects on pests. Especially sweet gum tree has provided away from the potato plant which was damaged because of the smell of spring. This plant should be done utilized after a purified active substance is isolated, essence of which we believe would be more effective in pest control. For use in the field of plants used in different areas will increase the interest in these plants. The use of sweet gum tree more carefully, which is considered an endemic plant and it would be good to expand.

ACKNOWLEDGEMENT Authors thank to Kemal YILMAZ for field application of two extracts, and Ufuk USLU for technical support at laboratory.

REFERENCES

Abramson, D., Goad, L. J., Goodwin, T. W., 1973, Triterpenes and sterols of Buxus sempervirens and local variations in their levels. Phytochemistry, 12: 2211-2216. Ash, M., Ash, I., 1995, Handbook of Food Additives: An international guide to more than 7000 products by trade name, chemical, function and manufacturer. Gower Publishing, Aldershot, 1040. Berenbaum, M., 1985, Brementtown revisited: Interactions among allelochemicals in plants. Recent Advances in Phytochemistry, 19: 139-169. Bianchi, F. J. J. A., Mikos, V., Brussaard, L., Delbaere, B., Pulleman, M. M., 2013, Opportunities and limitations for functional agrobiodiversity in the European context. Environmental Science & Policy, 27: 223-231. Boland, D. J., Brophy, J. J., House, A. P. N., 1991, Eucalyptus Leaf Oils: Use, Chemistry, Distillation and Marketing. Inkata Press, Melbourne, Australia, 252. Bowers, W, S., Ohta, T., Cleere, J. S., Marsella, P. A., 1976, Discovery of insect anti-Juvenile hormones in plants. Science, 193: 542-547. Davis, P. H., (1966-1988), Flora of Turkey and The East Aegean Islands, vol. 1-10. Edinburgh, Edinburgh University Press. De Groot, A. E., van Beek, T. A., 1987, The synthesis of Drimane and Clerodane insect antifeedants. Recueil des Travaux Chimiques Pays-Bas, 106: 1-18. Dorow, E., 1993, Present practices of controlling desert locust outbreaks. In: Rembold, H. (Ed.). New strategies for locust control, ATSAF, Bonn, 7-8. Ecevit, O., 1988, Zirai Mücadele İlaçları ve Çevreye Olan Etkileri, Samsun, Ondokuz Mayıs Üniversitesi Yayınları, 57 (in Turkish). Ertürk, Ö., 2002, Elma zararlıları Cydia pomonella ve Yponomeuta malinellus’un patoloji ve mikrobiyal kontrolleri, Doktora tezi, Karadeniz Teknik Üniversitesi Fen Bilimleri Enstitüsü, Trabzon, Türkiye (in Turkish). Ertürk, Ö., Uslu, U., 2007, Antifeedant, growth and toxic effects of some plant extracts on Leptinotarsa decemlineata (Say) (Cleoptera, Chrysomelidae). Fresenius Environmental Bulletin, 16: 601-607. FAO, 1992, Pesticide Residues in Food. Report No: 116, 146. Fenemore, P. G., 1984, Plant Pests and Their Control, Butterworths, London, U. K., 280. Franzen, H., 1993, Need for development of new strategies for locust control. In: Atsaf, R. H. (Ed.). New strategies for locust control, Bonn, 9-13. Gurbuz, I., Yesilada, E., Demirci, B., Sezik, E. A., Demirci, F., Baser, K. H. C., 2013, Characterization 111 Effects of Various Plant Extracts on the Development of the Potato

of volatiles and anti-ulcerogenic effect of Turkish sweetgum balsam (Styrax liquidus). Journal of Ethnopharmacology, 148: 332-336. Handal, A. J., Hund, L., Paez, M., Bear, S., Greenberg, C., Fenske, R. A., Barr, D. B., 2016, Characterization of pesticide exposure in a sample of pregnant women in Ecuador. Archives Environmental Contamination and Toxicology, 70(4): 627-39. Holopainen, M., Jahodar, L., Seppanen-Laakso, T., Laakso, I., Kauppinen, V., 1988, Antimicrobial activity of some Finnish Ericaceous plants. Acta Pharmaceutica Fennica, 97: 197-202. Isman, M. B., 2006, Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly world. Annual Review of Entomology, 51: 45-66. Lamparski, R., Wawrzyniak, M., 2005, Effects of water extracts from Geraniaceae plants with adjuvant added on feeding and development of Colorado potato beetle (Leptinotarsa decemlineata Say). Journal of Plant Protection Research, 45: 115-123. Lee, B. H., Annis, P. C., Tumaalii, F. A., Choi, W. S., 2004, Fumigant toxicity of essential oils from the Myrtaceae family and 1,8-Cineole against 3 major stored-grain insects. Journal of Stored Products Research, 40: 553-564. Matthews, G., 2006, Pesticides: Health, safety and the environment. 1st edn. Wiley-Blackwell Publishing, New York, U. S. A., 248. Mohan, C. G., Gokavi, S., Viswanatha, G.L., Shylaja, H., Nandakumar, K., 2009, Analgesic and antiinflammatory activity of leaf extracts of Commiphora caudata in Rodents. Pharmacologyonline, 2: 991-998. Mukherjee, S., 2003, Influence of plant allelochemicals on growth rate, nutritional physiology and mid-gut esterase activity in fifth instar larvae of Spodoptera litura (F.) (Lepidoptera: Noctuidae). Invertebrate Reproduction and Development, 43(2): 125-132. Nawrot, J., Bloszyk, E., Harmatha, J., Novotny, I., Drozdz, B., 1986, Action of antifeedants of plant origin on beetles infesting stored products. Acta Entomologica Bohemoslovaca, 83: 327-335. Norris, D. M., 1986, Anti-feeding compounds. In: Bowers, W. S., Ebing, W., Fukuto, T. R., Martin, R. W., Yamamoto, I. (Eds.). Chemistry of Plant Production Volume 1: Sterol Biosynthesis, Inhibitors and Anti-Feeding Compounds, Academic-Verlag, Berlin, 1-92. Palmquist, K., Fairbrother, A., Salatas, J., Guiney, P. D., 2011, Environmental fate of pyrethroids in urban and suburban stream sediments and the appropriateness of Hyalella azteca model in determining ecological risk. Integrated Environmental Assessment and Management, 7: 325-335. Rembold, H., 1994, Advances in invertebrate reproduction. Elsevier Science Publishers, 3: 481-491. Saxena, R. C., 1987, Antifeedants in tropical pest management. Insect Science and Application, 8: 731-736. Scott, I. M., Jensen, H., Scott, J. G., Isman, M. B., Arnason, J. T., Philogéne, B. J., 2003, Botanical insecticides for controlling agricultural pests: Piperamides and the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Archives Insect Biochemistry and Physiology, 54(4): 212-225. Smith, A. E., Secoy, D. M., 1975, Forerunners of pesticides in classical Greece and Rome. Journal of Agricultural and Food Chemistry, 23: 1050-1055. Soto, A., Venzon, M., Oliveira, R. M., Oliveira, H. G., Pallini, A., 2010, Alternative control of Tetranychus evansi Baker & Pritchard (Acari: Tetranychidae) on tomato plants grown in greenhouses. Neotropical Entomology, 39: 638-644. Thacker, J. R. M., 2002, An Introduction to Arthropod Pest Control. Cambridge Univ. Press. U. K., 337. Turkish Ministry of Agriculture and Rural Affairs, 2008. The technical guide of Agricultural control, T.C. Ankara, Turkey, 5: 110-112. Van Beek, T. A., De Groot, A. E., 1986, Terpenoid antifeedants. Part. I: An overview of terpenoid antifeedants of natural orgin. Recueil des Travaux Chimiques Pays-Bas, 105: 513-517. 112 ERTÜRK, Ö., SARIKAYA, A.

Ware, G. W., 1982, Pesticides: Theory and Application, W. H. Freeman and Company, New York, U. S. A., 308. Whittaker, R. H., Feeny, P. P., 1971, Allelochemics: Chemical interactions between species. Science, 171: 757-770. Wieczorek, M., 1996, The effects of plant extracts on the cabbage butterfly,Pieris brassicae (Lepidoptera). Polish Journal of Entomology, 65: 93-99. Yıldırım, A., Mavi, A., Oktay, M., Kara, A. A., Algur, O. F., Bilaloğlu, V., 2000, Comparison of antioxidant and antimicrobial activities of tilia (Tilia argentea Desf ex DC), sage (Salvia triloba I.), and black tea (Camellia sinensis) extracts. Journal of Agricultural and Food Chemistry, 48: 5030-5034. Zehnder, G., Gurr, G. M., Kühne, S., Wade, M. R., Wratten, S. D., Wyss, E., 2007, Arthropod pest management in organic crops. Annual Review of Entomology, 52: 57-80.

Received: May 09, 2016 Accepted: April 10, 2017 J. Entomol. Res. Soc., 19(2): 113-119, 2017 ISSN:1302-0250

A New Genus and Species of Aphodiini (Coleoptera: Aphodiidae) from Istanbul Turkey

Yakup ŞENYÜZ

Dumlupinar University, Faculty of Arts and Science, Department of Biology, Kütahya, TURKEY, e-mail: [email protected]

ABSTRACT Bosphorus bariscercii n. gen. and n. sp. is described and illustrated in Turkey (type locality: İstanbul, Esenyurt). The newly discovered genus mostly resembles the genus Bodilus Mulsant and Rey, 1870. Key words: Taxonomy, new genus, new species, Aphodiidae, Bosphorus, Turkey.

INTRODUCTION Family Aphodiidae is part of superfamily Scarabaeoidea and comprises approximately 358 genera and 3395 species worldwide (Roskov et al., 2016). There are 1084 species belonging to 155 genera from 6 tribes in the palaearctic region (Dellacasa et al., 2016) and 151 species and 4 subspecies belonging to 44 genera in the fauna of Turkey (Carpaneto et al., 2000; Dellacasa and Dellacasa (2006); Şenyüz, 2009; Rozner and Rozner, 2009). Some of researchers studied on this family thought that Aphodius has many subgenera (Schmidt, 1922; Balthasar, 1964). This matter was evaluated in the book of Dellacasa and Dellacasa (2006) edited by Löbl and Smetana. All the subgenus was accepted almost as genus in the revision on Dellacasa et al. (2001) and Dellacasa et al. (2016) edited by Löbl and Löbl. Taxon mentioned at the current literature should not be called subgenus but it can be named as genus. The new genus includes tribe Aphodiini. Since it has have different characteristics than other genera in the Aphodinae, the sample was the sample was identified as a new genus and new species.

MATERIAL AND METHOD The present work is based on a study of a specimen collected by Barış Çerçi and kept in the collection of the author in the Dumlupinar University Entomology Museum (DPUEM). The new species was described based on external morphology, epipharynx and male genitalia as described in Dellacasa et al. (2001). The samples were photographed by using Olympus SZX9 and Nikon AZ 100. 114 ŞENYÜZ, Y. RESULTS The main characteristics of the new genus are: Medium size species (length 9 mm), scutellum small, equilateral triangular with curved sides, blackish, aedeagus rather stout: paramera short, widened and membranous apically. Some of these characteristics are common in few genera of Aphodinae (Lunaphodius, Bodilus, Phalacronothus). However, some characteristics such as, totally blackish body, front legs more or less dark brownish, head with epistome rather convex at center with a tuberculiform curved transverse carina, irregularly punctured on disc, genae thinly bordered, there are points in 3 different sizes on the head (Fig. 1). Large punctures are scattered on the rear of the frontal suture (Fig. 2). Medium sized punctures predominant between frontal suture and epistome, the remaining gaps scattered with small punctures. Fore tibia distally tridentate and proximally not serrulate at outer margin; upper side smooth, apical spur truncate and obtusely inward hooked apically; First segment of hind tarsi as long as following two combined, shorter than superior apical spur. Pygidium with some discal and distal elongate setae. Aedeagus rather stout: paramera short, widened (Figs. 3, 4, 5). Epipharynx feebly sinuate at front magrin, more or less widely at sides; epitorma, drop-shaped, glabrous and broadened towards base; pedia with sparse prophobae, mixed many stout equal size chaetae; corypha with two elongate, strongly prominent celtes; chaetopariae, short, densely and serially arranged; tormae rather short (Fig. 6), are specific for this new genus.

1 2

Figs. 1, 2. Bosphorus bariscercii n. gen., n. sp., 1. Three different sizes punctures on the head in same pictures. 2. Near of the front.

3 4 5

Figs. 3, 4, 5. Bosphorus bariscercii n. gen., n. sp., aedeagus: 3 dorsal view. 4 lateral view. 5 ventral view. 115 A New Genus and Species of Aphodiini

Fig. 6. Bosphorus bariscercii n. gen., n. sp., epipharynx.

Bosphorus gen. n. Type species: Bosphorus bariscercii sp. n. Etymology: There is a longitudinal carina on the head, which resembles the genus Bosphorus. In addition the type specimen was collected near the Bosphorus in Istanbul, Turkey. Description: Medium size species (length 9 mm), Blackish. Head with epistome rather convex at center with a tuberculiform curved transverse carina; frontal suture trituberculate, there is longitudinal carina between median tubercle of frontal suture and tuberculiform of the epistome. Fore tibia distally tridentate and proximally not serrulate at outer margin; upper side smooth, apical spur truncate and obtusely inward hooked apically; Middle and hind tibiae normally widened toward apex and with strong transverse carinae on outer face, apically fimbriate with spinules unequal and irregularly elongated. Pronotum convex, transverse, shiny; Scutellum small, equilateral triangular with curved sides. Elytra elongated, convex, striated. Aedeagus rather stout, paramera short, widened. Distribution: Turkey Bosphorus bariscercii sp. n. (Figs. 1-15) Type locality: Turkey, İstanbul, Esenyurt. Type series: Holotype 1 ♂. Turkey, İstanbul, Esenyurt., 50 m, 02.02.2015, leg. B. Çerçi (DPUEM). Type depository. Holotype: in Dumlupinar University Entomology Museum (DPUEM). Type labelling. Holotype with two labels: 1st, on the white paper, printed in black: “İstanbul; Esenyurt 50 mt.; 01.02.2015; leg. Çerçi, B. 2nd, on the red label, printed in black: “Holotype; Bosphorus; bariscercii; Y. Şenyüz, 2015”. Etymology: The species is dedicated to Barış Çerçi, who collected the holotype. Description: Lenght 9 mm: rather stout, nearly glabrous, elongated, convex, shiny (Figs. 7, 8); Blackish. Front legs more or less dark brownish. Head with epistome rather convex at center with a tuberculiform curved transverse carina, irregularly punctured on disc, punctuation more supercicial distally; clypeus feebly sinuate at middle, widely rounded at sides, margins shortly and sparsely bristled, genae thinly bordered, obtusely 116 ŞENYÜZ, Y. rounded, ciliate, protruding from the eyes; latter medium sized (Fig. 9); frontal suture trituberculate, there is longitudinal carina between median tubercle of frontal suture and tuberculiform epistome (Figs. 9, 10); There are points in 3 different sizes on the head (Fig. 1). The smallest of them are like tiny holes. Medium-sized ones are twice as large as small ones. The biggest ones are 5 times bigger than the small ones and 2.5 times bigger than the medium ones. Large punctures are scattered on the rear of the frontal suture (Fig. 2). Medium sized punctures predominant between frontal suture and epistome, the remaining gaps scattered with small punctures. Pronotum convex, transverse, shiny with doubly punctured (Fig. 11), throughout scattered small punctures, larger punctures sparse on disc and more dense at sides, sides quite bordered, hind angels obtusely rounded, visible from above, not reflexed downwards; base bisinuate, quite bordered, middle very shortly bristled. Scutellum small, its long average 1/13 length of elytra at suture (Fig. 7), equilateral triangular with curved sides (Fig. 12), with few punctures on basal half. Elytra distinctly pubescent on sides and apically (x50), elongated, convex, striated, shiny, fine and deep; interstices nearly flat, rather dull, usually strongly microreticulate and distinctly punctured. Elytra with 10 striae, (Fig. 13). Fourth and sixth elytral interstices joined preapically (Fig. 13). Metasternal plate sunk, shiny, microreticulate, longitudially with distinctly grooved, glabrous, marginal punctures with longer, backwards seta. Abdominal sternites, Fore tibia distally tridentate and proximally not serrulate at outer margin (Fig. 14); upper side smooth, apical spur truncate and obtusely inward hooked apically; Middle and hind tibiae normally widened toward apex and with strong transverse carinae on outer face, apically fimbriate with spinules unequal and irregularly elongated (Fig. 15). First segment of hind tarsi as long as following two combined, shorter than superior apical spur. Pygidium with some discal and distal elongate setae. Aedeagus rather stout: paramera short, widened (Figs. 3, 4, 5). Epipharynx feebly sinuate at front margin, more or less widely at sides; epitorma, drop-shaped, glabrous and broadened towards base; pedia with sparse prophobae, mixed many stout equal size chaetae; corypha with two elongate, strongly prominent celtes; chaetopariae, short, densely and serially arranged; tormae rather short (Fig. 6).

Figs. 7, 8. Bosphorus bariscercii n. gen., n. sp. 7. Dorsal view 8. Ventral view. 117 A New Genus and Species of Aphodiini Distribution: The new species is known only from the type locality. Female: Unknown

Fig. 9. Bosphorus bariscercii n. gen., n. sp., dorsal view of head.

10 11 12

Figs. 10, 11, 12. Bosphorus bariscercii n. gen., n. sp. 10. The sunken longitudinal carina between median tubercle of frontal suture and tuberculiform epistome.11. Dorsal view of the base of. pronotum with double punctation. 12. Scutellum.

Fig. 13. Bosphorus bariscercii n. gen., n. sp., the seventh and ninth elytral interstices joined preapically.

14 15

Figs. 14, 15. Bosphorus bariscercii n. gen., n. sp., 14. Left front leg (dorsal view) 15. apex of hind tibia. 118 ŞENYÜZ, Y. DISCUSSION Some external morphological characteristics of this new genus such as the clypeus feebly sinuate at middle, rounded at sides; genae obtusely rounded, elongately bristled, protruding than eyes; latter medium size; frontal suture distinct, trituberculate; Scutellum small, triangular, microreticulate, usually punctured on basal half, at most as long as 1/7 of sutural margin of elytra or shorter; Pronotum not bordered at front margin, base of pronotum bordered; Hind tibiae apically fimbriate with more or less elongate and irregularly; Elytra more or less distinctly pubescent at least on sides or apically (x50); Aedeagus digitiform, is similar to Bodilus (partim; lugens group) Mulsant and Rey, 1870 genera. In this instance the longitudinal carina between median tubercle of frontal suture and tuberculiform epistome of the species can be used as a fundamental characteristic of the new genus, since it has been observed that the sample does not match the determination key item 121 proposed by Dellacasa et al. (2001). At this stage, the mentioned two genera are Lunaphodius Balthasar, 1964, and Bodilus Mulsant and Rey, 1870. The basic morphological characteristic of holotype that does not comply with either genera with longitudinal carina between median tubercle of frontal suture and tuberculiform epistome. The color is the second important distinguishing character. The holotype is completely black while both of the mentioned genera have different colors. Male genitalia resembles general structure of genus Phalacronothus Motschulsky, 1859. However, this genus is quite different in both character and its size varies between 2.5-4 mm. Another important characteristic feature is the existence of punctures of three different sizes in different regions around the epistome and frontal suture. The final difference is the aedeagus. Supplement to the key in Dellacasa et al. (2001) 121 bis (120) - Rather stout, elongated, convex, shiny. Blackish. Head with epistome rather convex at center with a tuberculiform curved transverse carina, genae thinly bordered, frontal suture trituberculate, there is longitudinal carina between median tubercle of frontal suture and tuberculiform epistome. Lenght 9 mm, European part of the Turkey …………………………………………..……………..…………Bosphorus 121 bis’ - Not all of the above characters simultaneously present ………………. 122 122 bis (121) - Genae quite rounded, feebly more protruding than eyes (figs 217, 219); sexual dimorphism accentuate: in males, clypeus more or less strongly sinuate at middle and apical spur of fore tibiae inserted at middle of inner margin (fig. 218). Blackish; elytra yellowish with brownish suture or blackish with wide humeral spot and apex reddish. Length 6-8 mm. Central Asia ...... Lunaphodius 122 bis’ - Genae obtusely angulate, distinctly more protruding than eyes (fig. 221); sexual dimorphism relatively feeble: in males, clypeus feebly sinuate at middle as in females, apical spur of fore tibiae inserted at inner margin at level of second outer distal tooth. Reddish testaceous or piceous; sides of pronotum and elytra testaceous or yellowish, sometimes latter with large cloudy discal spot brownish. Length 5-12 mm. Palearctic, Afrotropical, and Nearctic (?) region ...... Bodilus (partim: lugens group) 119 A New Genus and Species of Aphodiini REFERENCES

Balthasar, V., 1964, Monographie der Scarabaeidae und Aphodiidae der palaeartischen und orientalischen region. Verlag der Tschechoslowaischen Akademie der Wissenschaften Prag, 3: 654 p. Carpaneto, G. M., Piattella, E., Pittino, R., 2000, The Scarab beetles of Turkey: An updated checklist and chorotype analysis (Coleoptera, Scarabaeoidea). Biogeographia, 21: 217-240. Dellacasa, G., Bordat, P., Dellacasa, M., 2001, A revisional essay of world genus-group taxa of Aphodiinae (Coleoptera: Aphodiidae). Memorie della Società Entomologica Italiana, 79: 1-482. Dellacasa, G., Dellacasa, M., Král, D., Bezděk, A., 2016, Tribe Aphodiini Leach, 1815. In: Löbl I. and Löbl D. (Eds.); Catalogue of Palaearctic Coleoptera, Scarabaeoidea, Scirtoidea, Dascilloidea, Buprestoidea, Byrrhoidea. Revised and Updated Edition. Brill, Leiden/Boston, 3: 98-155 Dellacasa, M., Dellacasa, G., 2006, Tribe Aphodiini Leach, In: Löbl I. and Smetana A. (eds.). Catalogue of Palearctic Coleoptera. Stenstrup: Apollo Books, 3: 105-142 Roskov Y., Abucay L., Orrell, T., Nicolson D., Flann C., Bailly N., Kirk P., Bourgoin T., DeWalt R. E., Decock W., De Wever A., eds., 2016, Species 2000 and ITIS Catalogue of Life, 2016 Annual Checklist. Digital resource at www.catalogueoflife.org/annual-checklist/2016. Species 2000: Naturalis, Leiden, the Netherlands. ISSN 2405-884X. Rozner, I., Rozner, G., 2009, Additional data to the Lamellicornia Fauna of Turkey (Coleoptera: Lamellicornia), Natura Somogyiensis, 15: 69-100. Schmidt, A., 1922, Coleoptera Aphodiinae. Das Tierreich. Lieferung, Walter de Gruyter Berlin und Leipzig: 45: 614 pp. Şenyüz, Y., 2009, Fauna of Subfamily Aphodiinae (Scarabaeidae, Coleoptera) in Turkmen Mountain, Dumlupinar University, Institute of Science And Technology, Department of Biology, PhD thesis, 90 pp.

Received: August 10, 2016 Accepted: June 28, 2017 AUTHOR GUIDELINES Journal of the Entomological Research Society (J. Entomol. Res. Soc.) accepts and publishes original research articles in the all field of entomology. The journal publishes regular research papers, review articles. Short, timely reports may be submitted as short communications. The Editors first evaluate all manuscripts. At this stage insufficiently original, have serious scientific flaws, have poor grammar or English language, or are outside the aims and scope of the journal papers will be rejected. Those that meet the minimum criteria are passed on to at least 2 experts for review. Authors of manuscripts rejected at this stage will usually be informed at most 1 months of receipt. Authors should suggest ([email protected]) four reviewer name, address and e-mail address about manuscript subject to examine the manuscript. At most two of them will be in your country and others will be different countries. Two reviewers are selected from these or editor assign another reviewers. A final decision to accept or reject the manuscript will be sent to the author along with any recommendations made by reviewers. Editor’s Decision is final Reviewers advise the editors, who are responsible for the final decision to accept or reject the article. Manuscript must be written in Arial with 12 type size with double spacing in Word for Windows. Please do not make paragraph in the text. Manuscripts generally should not exceed 30 pages.

Heading: The title of the paper should be informative, but preferably not exceed twenty words. Just under the heading, give title, full name(s) of author(s) (surname(s) in capital letters), with full address and e-mails of each on a separate line.

Abstract: An abstract provided at the beginning of the paper will indicate the main aspects of the subject, to be followed by 5-7 key words.

Text: The standard order of sections for original papers is as follows: Introduction, Material and Methods, Results, Conclusions and Discussion, Acknowledgements, References. The scientific names (e.g. genus- and species-group names) are to be italicized. For faunistic research follow this order, Distribution:.., Material examined:…, Host plant:…. Example: Sphex oxianus Gussakovskij, 1928 Distribution: Central and South West Asia, Afghanistan, Iran, Israel, Turkey (Bohart and Menke, 1976; Menke and Pulawski, 2000; Kazenas, 2001), Turkey: Artvin (De Beaumont, 1967). Material examined: Ankara, Altındağ, Çubuk Dam Lake, 900 m, 29. 07. 1998, 1 ♂; Kalecik, 600 m, 24. 06. 2001, 2 ♀♀. Host plant: Echinophora sp. Please use ♀, ♂ symbols. Please write upper genus categories with capital letters.

Illustrations: Illustrations, graphs, their caption or legends should form separate, self- explanatory unit. Abbreviations in the legends should be explained but if there are too many, they should be included into a separate list. The original drawing and photographs should be not more than twice as large as when printed. Morphological illustrations should be including a scale bar. Photographs and electron micrographs must be in JPEG file format (300 dpi). Drawings (black and white type) must be in TIFF format and their size no more than 10 MB. Graphs should be in excel file format. Color figures pay charge. Tables should include headings and explanations, and should be numbered consecutively. Their approximate position in the text should be indicated in the margin. Legends and title of the graphs and tables must be in Arial with 12 type size. Please do not embed the figures, graphs and table into the text, send them as supplementary files. In the text attribution to the figures should be given in parenthesis and must be abbreviate like this; (Fig.1).

References: In the text, reference to the literature should conform to the “name- and- date” system, e.g. (Lyneborg, 1983); Beirne and Young (1953); Edwards (1894-1896); Gayuboet al. (2003). Titles of papers published in languages other than the major ones (English, German, French, Spanish, Portuguese, Turkish) should be an English translation (in parentheses) with an explanatory note at end, e.g. (in Russian). The list references will be given at the end of the article and listed alphabetically, according to the following examples, all periodicals must be unabbreviated and italicized.

Journal Article Beirne, B. P., Young, D. A., 1953, The North American species of Cicadula (Homoptera, Cicadellidae). Canadian Entomologist, 85: 215-226.

Book Chapter Kirejtshuk, A. G., 1992, Evolution of mode of life as the basis for division of the beetles into groups of high taxonomic rank. In: Zunino, M., Bellés, X., Blas, M. (Eds.). Advances in Coleopterology. European Association of Coleopterology, Barcelona, 249-261.

Book Steinmann, H., Zombori, L., 1985, An Atlas of Insect Morphology. 2nd edn. Akadèmiai Kiadò, Budapest, Hungary, 253.

URLs Geller-Grimm, F., 2007, Database Asilidae: Catalog of Species. http://www.geller-grimm.de/ catalog/species.htm (10.03.2010). Nomenclature must be in absolute agreement with current ICZN rules. The only acceptable type concepts are: holotype, paratype etc. The following abbreviations should be adopted: gen. n., sp. n., stat. n. and comb. n. 10 free reprints are supplied per contribution: An additional number may be ordered at the prices quoted on the order form sent to the corresponding author. Journal of the Entomological Research Society uses the Open Journal Systems (OJS) platform, which will enable the journal to accept submissions online. For submitting a manuscript please go to web page http://www.entomol.org and register as author and submit your manuscript online. Manuscripts send by e-mail will be not accepted. URL: http://www.entomol.org e-mails: [email protected]; [email protected]

Address: Journal of the Entomological Research Society, P.box.110 Bahcelievler P.Isl.Mud. 06502, Ankara/TURKEY CONTENTS

JING, T., HU, X., HU, C., LI, C., LIU K., WANG, Z., Life Cycle and Host Preference of Xylotrechus rusticus (L., 1758) (Coleoptera: Cerambycidae) in the Heilongjiang Province, China ...... 1

XUE, W., YU, T., Review of the Phaonia consobrina Group (Diptera: Muscidae) from China, with Descrip- tions of Three New Species ...... 11

HOLUŠA, J., LUKÁŠOVÁ, K., Pheromone Lures: Easy Way to Detect Trypodendron Species (Coleop- tera: Curculionidae) ...... 23

WAGNER, L. S., FENOGLIO, M. S., SALVO, A., Alien Species Numerically Dominate Natural Enemy Communities in Urban Habitats: A Preliminary Study ...... 31

TSUKADA, M., INUI, M., SUZAKI, N., Do Beetles Prefer the Odor of Female-Stage to Male-Stage Flow- ers in Atemoya, a Cantharophylous Protogynous Fruit Tree (Annonaceae)? ...... 43

MAHMOUD, E. A., GABARTY, A., . Impact of Gamma Radiation on Male Proboscis of Rhynchophorous ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae)...... 53

JAPOSHVILI, G., KOSTJUKOV, V., KOSHELEVA, O., YEGORENKOVA, E., New Record of Neotrichopo- roides (Hymenoptera: Eulophidae) from Georgia with some Taxonomic and Biogeographical Notes ...... 67

VESNIC, A., Rifat SKRIJELJ, R., Sadbera TROZIC-BOROVAC, S., TOMANOVIC, Z., Diversity and Nesting Preferences of Camponotus lateralis Group Species on Western Balkan Peninsula (Hymenoptera: Formicidae) ...... 73

ÇELİK, D., ÖZBEK, R., UÇKAN, U. Effects of Indole-3-Acetic Acid on Hemocytes of Achoria grisella Fabr. (Lepidoptera: Pyralidae) ...... 83

JAPOSHVILI, G., KOSTJUKOV, V., Three New Minotetrastichus (Hymenoptera: Eulophidae) Species Records from Transcaucasia ...... 95

ERTÜRK, Ö., SARIKAYA, A., Effects of Various Plant Extracts on the Development of the Potato Beetle under Laboratory and Field Conditions: A Combined Study ...... 102

ŞENYÜZ, Y., A New Genus and Species of Aphodiini (Coleoptera: Aphodiidae) from Istanbul Turkey ....113