The Role of Turnera Subulata and Cosmos Sulphureus Flowers in the Life of Anagrus Nilaparvatae (Hymenoptera: Mymaridae)

Anagrus nilaparvatae is a potential egg parasitoid to control the rice brown planthopper (Nilaparvata lugens Stal.) The parasitoid needs to consume suitable food to maximize its biotic potential and parasitizing ability. This study was aimed to determine the benefits provided by the presence of Turnera subulata and Cosmos sulphureus flowers on the life of A. nilaparvatae. This study consisted of two experiments. The first experiment was designed to determine the effects of the tested flowers on the parasitism and hatching rate of A. nilaparvatae on N. lugens eggs. The flowers were set inside the rearing cage of parasitoid in the presence of N. lugens eggs in Ciherang rice seedlings. In addition, honey and the control (no feed added) were included into the treatments, totalling of four treatments. The parasitism rate of A. nilaparvatae fed with the flowers or honey was similar to those unfed. However, the hatching rate of A. nilaparvatae was much higher on those fed with flower or honey than those unfed. The number of A. nilaparvatae unable to eclose from eggs of N. lugens for the unfed parasitoid was 37.4% in comparison with 8.19 to 15.67% for those fed with flower or honey. The second experiment was a follow-up to address the question on the fitness of progeny of A. nilaparvatae fed with the tested flowers. The flowers and honey did not increase the longevity of A. nilaparvatae progeny. However, A. nilaparvatae fed with flowers or honey produced progeny that resulted in higher number of offspring compared to those unfed. C. sulphureus flower significantly increased the number of offspring produced by A. nilaparvatae. This suggest that the diet of the parental parasitoid determines the fitness of the progeny. Improving the hatching rate and the fecundity of progeny produced by the adults of A. nilaparvatae fed with the flower of T. subulata and C. sulphureus would contribute to the increasing population of this parasitoid which could lead to a better control of N. lugens in the rice field. IntisariAnagrus nilaparvatae merupakan parasitoid telur yang potensial untuk mengendalikan serangan wereng batang cokelat (Nilaparvata lugens Stal.). Parasitoid perlu mendapatkan sumber pakan yang sesuai untuk memaksimalkan potensi biologis dan kemampuan memarasit inang. Penelitian ini bertujuan untuk mengkaji manfaat pemberian bunga Turnera subulata dan Cosmos sulphureus pada kehidupan A. nilaparvatae. Penelitian ini terdiri dari dua percobaan. Percobaan pertama dirancang untuk mengetahui pengaruh bunga yang diuji terhadap tingkat parasitasi dan penetasan telur A. nilaparvatae pada telur N. lugens. Bunga diletakkan di dalam tabung rearing parasitoid yang di dalamnya terdapat bibit padi varietas Ciherang yang mengandung telur N. lugens. Selain itu, terdapat pula perlakuan madu dan kontrol (tanpa bunga dan tanpa madu), dengan total empat perlakuan. A. nilaparvate yang diberi pakan bunga atau madu memiliki tingkat parasitasi yang sama dengan yang tidak diberi pakan. Namun, tingkat penetasan telur A. nilaparvatae yang diberi pakan bunga atau madu jauh lebih tinggi daripada yang tidak diberi pakan. Jumlah A. nilaparvatae yang tidak menetas dari telur N. lugens pada parasitoid yang tidak diberi pakan adalah 37,4% dibandingkan dengan parasitoid yang diberi pakan bunga atau madu yang berkisar 8,19 - 15,67%. Percobaan kedua adalah tindak lanjut untuk menjawab pertanyaan tentang kebugaran keturunan A. nilaparvatae yang diberi pakan dengan bunga yang diuji. Bunga dan madu tidak meningkatkan lama hidup keturunan A. nilaparvatae. Namun, A. nilaparvatae yang diberi pakan dengan bunga atau madu menghasilkan keturunan yang memiliki fekunditas lebih tinggi dibandingkan dengan keturunan dari A. nilaparvatae yang tidak diberi pakan. Bunga C. sulphureus secara signifikan mampu meningkatkan jumlah keturunan yang dihasilkan oleh A. nilaparvatae. Ini menunjukkan bahwa jenis pakan induk parasitoid menentukan kebugaran keturunannya. Peningkatan penetasan dan fekunditas dari keturunan yang dihasilkan oleh induk A. nilaparvatae yang diberi pakan bunga T. subulata dan C. sulphureus akan berkontribusi pada peningkatan populasi parasitoid sehingga dapat mengendalikan serangan N. lugens di pertanaman padi dengan lebih baik.


INTRODUCTION
The rice brown planthopper (Nilaparvata lugens Stal.) (Hemiptera: Delphacidae) is a major pest of rice plants in Asia with the damage could reach 90% (Liu et al., 2012). This pest is a serious threat in rice production because it may cause hopperburn (Catindig et al., 2009), and vector for Rice grassy stunt virus (RGSV) and Rice ragged stunt virus (RRSV) (Reissig et al., 1986).
Parasitoids have been negatively impacted by the use of chemical pesticides, starting from reduction in their fitness and parasitizing ability to their death (Hardin et al., 1995;Meilin et al., 2012a). Improvement in the role of parasitoids as Biological Control Agents (BCA) can be done through ecological engineering so that optimized ecosystem services may result in a significant control of N. lugens. The existence of parasitoids can be preserved by increasing the biodiversity of rice ecosystems. Planting flowering plants (non-crop plants) around the crops will be able to provide a source of food (pollens and nectars), a shelter, as well as alternative hosts for parasitoids (Wratten et al., 2003).
Several previous studies have shown that addition of flowering plants could increase insect fecundity and longevity (Pianka et al., 1977;Wratten et al., 2003;Berndt and Wratten, 2005;Abd El-Kareim et al., 2011). Zhang et al. (2004) reported that the longevity and fecundity of Trichogramma brassicae increased after feeding the parasitoid with honey and corn pollen. A. nilaparvatae and A. optabilis fed with sesame flowers had longer life and better parasitizing ability (Zhu et al., 2013). The longevity of A. nilaparvatae also became longer after being treated with the flowers of Phacelia tanacetifolia (Farrell, 2013). Egg parasitoids, including A. nilaparvatae, could control the population of N. lugens in the early stage and it made these parasitoids into potential BCA (Godfray, 1994). Adding flowering plants surrounding rice field could reduce insecticide applications by 70%, increased grain yield by 5%, and delivered an economic advantage of 7.5% (Gurr et al., 2016).
This study aimed to assess the effects of Turnera subulata and Cosmos sulphureus flowers on the parasitizing capacity of A. nilaparvatae, as indicated by the parasitism rate, parasitoid emergence, and the longevity and fecundity of parasitoid progeny after their parents were fed with these two flowers. T. subulata and C. sulphureus are widely adapted and planted crops and recently used by farmers as refuge in the rice fields in the Special Province of Yogyakarta and the Province of Central Java. In addition, T. subulata was selected to represent perennial flowering plants, while C. sulphureus represented seasonal flowering plants.

Rearing of Nilaparvata lugens
The starting population of N. lugens was collected from the rice field in Ngestiharjo Village, County of Kasihan, the District of Bantul, Yogyakarta and reared in the greenhouse of Crop Protection Department, Faculty of Agriculture, Universitas Gadjah Mada (UGM). N. lugens was reared on Ciherang rice variety employing an established procedure at the Laboratory of Pesticide Toxicology, UGM.
Kata kunci: Anagrus nilaparvatae, Cosmos sulphureus, Nilaparvata lugens, parasitasi, Turnera subulata Tanjungharjo Village, Nanggulan County, the District of Kulon Progo, Yogyakarta in November 2016. The rice variety used for trapping was Ciherang (one-month old) and this variety was similar to the rice field where the trapped crops were placed. Rice plants were grown in plastic pots with a diameter of 14 cm (top) and 11 cm (bottom), and 12 cm tall. Potted rice plants were individually covered with a plasic tube (50 cm tall, 10 cm diameter) constructed from mica plastic sheet. The tops of the tubes were covered with cotton gauze. Plants were infested with 50 gravid females of N. lugens and incubated for three days for oviposition. Subsequently, N. lugens and the plastic tube were removed. Rice plants contained with eggs of N. lugens were transferred to one month-old rice plants and placed randomly. Plants were left in the rice field for three days. Subsequently, leaves and roots of the plants were removed and left only the stems. The cut end of each stem was wrapped in tissue paper moistened with water. The stems were inserted in a plastic cup with 8 cm in diameter (top), 5.5 cm in the bottom, and 12 cm tall. Each plastic cup with rice stems were then placed inside a plastic tube (8 cm diameter, 25 cm tall). The plastic tubes were covered with cotton gauze on the top. The newly emerged A. nilaparvatae was transferred and reared in the Laboratory using established laboratory procedure (Meilin et al., 2012b) on Ciherang rice variety.

Flowers of Turnera subulata and Cosmos sulphureus
Turnera subulata and C. sulphureus were obtained from Banyumas, Central Java. T. subulata was propagated by cuttings and C. sulphureus was propagated by sowing the dried seeds in polybag of 30×30 cm in size. Flowers at the full bloom were used for the testing.

Effect of Turnera subulata and Cosmos sulphureus on Parasistism of Anagrus nilaparvatae
The experiment employed a Complete Randomized Design (CRD) with four treatments: T. subulata and C. sulphureus flowers, honey, and the control (no flower and no honey). The rice plant used was twoweeks old of Ciherang variety.
Ten rice seedlings were placed in plastic cups (6.5 cm top diameter, 4.5 cm bottom diameter, 4.5 cm tall). The cup was then placed inside a plastic tube (8 cm diameter, 23 cm tall) and the plants were maintained their freshness by adding water into the cup. Twenty N. lugens gravid females were released in the plastic tube for two days for oviposition when the adults were then removed. One flower of T. subulata or C. sulphureus was placed into each cup inside the plastic tube. For the treatment of honey, 10% honey solution (Madu Murni Nusantara) was smeared on plastic tube wall. The honey and control treatments were applied in the absence of flowers. Five A. nilaparvatae females were transferred into each plastic tubes set for the four different treatments. The flowers were replaced every other day until all the parasitoids died. Subsequently, the leaves of rice seedlings were removed and the roots were wrapped with tissue of 5×5 cm, moistened with water and wrapped in aluminum foil on the outside and placed into a test tube, and covered with cotton gauze. Each treatment was replicated 6 times.
Observation was conducted everyday by counting the number of the newly emerged A. nilaparvatae and N. lugens nymphs. After three days in a row without any emergence, the rice seedlings were dissected to record the number of N. lugens eggs being parasitized but not hatching (unhatched parasitized eggs) and the number of healthy (not parasitized) N. lugens eggs but not hatching. Parasitism rate was calculated by dividing the sum of emerged and died parasitoids inside the host by the sum of unhatched eggs, N. lugens nymphs and the newly emerged A. nilaparvatae. Analysis of Variance (ANOVA) was done using SAS 9.3, and was continued with LSD (P < 0.05) when significant differences existed (Gomez & Gomez, 1995).

Effect of Turnera subulata and Cosmos sulphureus on the Progeny of Anagrus nilaparvatae
The previous study was designed to determine the direct effects of T. subulata and C. sulphureus flowers on A. nilaparvatae (F 0 ). This study was the continuation of the previous one to address the questions whether the adults of A. nilaparvatae fed with the flowers produced better progeny (F 1 ) than those of unfed parasitoid. The design of this study was similar to the previous one, consisting of four treatments. A. nilaparvatae emerged from the all treatments were used according to their respective treatments. However, no matter from what treatments the parasitoids came from they were not fed with any materials (flower or honey). Therefore, it was expected that if the flowers had something good for the parent, the progeny would be beneficially affected by the life of their parents.
The rice plant used was Ciherang aged of two weeks. Five rice seedlings were placed in a glass vial (3.5 cm in diameter, 4.5 cm tall). The vial was then placed inside a plastic tube (5 cm diameter, 17 cm tall) and the plants were maintained their freshness by adding water into the vial. Five N. lugens gravid females were released in the plastic tube. One day after N. lugens release, one A. nilaparvatae adult was released into the plastic tube without food until the parasitoid insidedied . Subsequently, the leaves of rice seedlings were removed. The roots were wrapped in tissue of 5×5 cm, moistened with water followed by the second wrapping using aluminum foil on the outer side. The rice stems were placed inside the test tubes (one treatment in one tube) and covered with cotton gauze. Each treatment was replicated 20 times.
Observation was conducted everyday to determine the longevity and fecundity of A. nilaparvatae progeny. The longevity of A. nilaparvatae was calculated by summing up the number of days since adult emergence until died. Fecundity of the progeny was counted based on the number parasitoid emerged from the parents being treated with no food in this experiment added with the number of N. lugens eggs being parasitized but not hatching (unemerged parasitoid). Rice stems were dissected three days after the last parasitoid had emerged to count the number of unemerged A. nilaparvatae.
Sin -1 √X transformation was performed to normalize the data distribution. ANOVA test was performed using SAS 9.3, and was continued with LSD test (P < 0.05) when significant differences existed (Gomez and Gomez, 1995).

Effect of Turnera subulata and Cosmos sulphureus on Parasistism of Anagrus nilaparvatae
Addition of T. subulata and C. sulphureus flowers or honey had no effect on the parasitism rate of A. nilaparvatae on N. lugens eggs (Figure 1). This result might be due to the fact that A. nilaparvatae is pro-ovogenic nature which does not require food source for eggs production but energy is needed for the host-searching process and survival (English-Loeb et al. 2003;Farrell, 2013). Gravid female of A. nilaparvatae could immediately oviposit when she found a host (Lou et al., 2014). Furthermore, A. nilaparvatae might also collect carbohydrates from the honeydew excreted by N. lugens (Lee et al., 2006;Wackers et al., 2008), although in smaller amounts.
In contrast to the data on parasitism rate, parent parasitoids not receiving any food (control) resulted to high number of unsuccesful parasitoid progeny to emerge from N. lugens eggs. Close to 40% of parasitoid were not able to hatch when the parents were not fed with flowers or honey, while those fed with flowers or honey caused significantly lower number of unsuccesfull hatching (Figure 2). Furthermore, differences between the tested flowers and honey was not significant. Nectar or honey could provide additional capacity of parasitoids in storing glycogen (Olson et al., 2000), slowing the declining rate of lipids (Lee et al., 2004), and increasing the egg maturation (Tylianakis et al., 2004). Pro-ovogenic parasitoids can resorb eggs and divert their energy for host searching process (Rivero & Casas, 1999). These results indicate that providing flowers and honey could improve the nilaparvatae (F 0 ) were released in the cage containing rice plants infected by N. lugens eggs and the flower quality A. nilaparvatae eggs. High parasitism rate and high success of parasitoid emergence from the host would contribute to high population of parasitoid for the following generation which in turn could lead to provide a better control for N. lugens. The emergence of A. nilaparvatae exposed to T. subulata and C. sulphureus flowers started at the 11 th day and ended at the 15 th day after infestation. However, the emergence of parasitoids from the control and honey treatments occured one day after those fed with flowers and ended at the same time. The peak of emergence of A. nilaparvatae occurred at the 12 th and 13 th day (Figure 3). Haryati (2016) reported that A. nilaparvatae fed with honey started to emerge at the 12 th to 14 th day. The early emergence of A. nilaparvatae in the treatments of T. subulata and C. sulphureus flowers indicates that nutrients from the flowers may be able to optimize the level of vitellogenin (a precursor protein of yolk) (Zhang et al., 2016) which then increasing the maturation of eggs (Tylianakis et al., 2004). Thus, eggs hatch quickly and accelerate the process of parasitization.

Effect of Turnera subulata and Cosmos sulphureus on the progeny of Anagrus nilaparvatae
The addition of flowers or honey for the diet of parents parasitoids had no effect on the longevity of A. nilarpavatae progeny. However, it significantly increased the number of parasitoids produced by the progeny (Table 1). In other words, when the parents of A. nilaparvatae were fed with flower of C. sulphureaus, their progeny would resulted more offsprings than those not receiving any food supplement. The absence of any positive effects on the longevity of F 1 progeny might be related to the fact that females would die immediately after laying their last eggs and they did not spend time or energy on activities that did not contribute to their fitness (Segoli, 2013). On the other hand, the flower of C. sulphureus consumed by F 0 parent had its effect on the fecundity of F 1 progeny. These results confirm that food sources from flowers (pollen and nectar), which contains sugar, protein, amino acids, and lipids, could increase the fecundity of parasitoids, such as A. nilaparvatae (Baggen & Gurr, 1998;English-Loeb et al., 2003;Vattala et al., 2006;Sivinski et al., 2011;Zhu et al., 2013).
Turnera subulata and C. sulphureus flowers had different effects which likely due to differences in morphology, composition of nectar and pollen, as well as volatiles (Farrell, 2013). The most important morphological factor in the suitability of flowers is corolla width and depth, in relation to parasitoid head width and mouthpart structure. Previous research reported that Phacelia tanacetifolia flowers had no effect on the longevity of A. nilaparvatae (Farrell, 2013), even decreasing the longevity of Hymenopteran parasitoid than that of water (Vattala et al., 2006). Biological clock of T. subulata (the flower blooms at 7−8 a.m. and closes at 11−12 a.m.) is one of the biological factors that may limit the access of the parasitoid to its food sources. This result confirms that different species of flowering plant were associated with different families of parasitoids (Sivinski, 2011). Each flowering plant has variability in the attractiveness and suitability for certain parasitoid species (Farrell, 2013). This information is important for selecting the suitable flowering plants to enhance the role of parasitoids as BCA in agricultural ecosystems. Parasitizing ability of parasitoids in an agroecosystem will increase following the increase in fecundity, egg quality, longevity, and the acceleration of the emergence. The stability of sustainable parasitoid population must always be managed to optimize the ecosystem services (Altieri, 1999). The presence of flowering plants (non-crop plants) on crop cultivation is one of the natural enemies' conservation and contribution of biodiversity to optimize the ecosystem services (Gurr et al., 2016).

CONCLUSION
The presence of T. subulata and C. sulphureus flower improved the hatchability of A. nilaparvatae from N. lugens eggs but they did not affect the parasitism rate. Under laboratory condition, the progeny of A. nilaparvatae produced from the parents reared with the flowers emerged one day earlier than those not receiving any food supplement. Although their progeny had the same longevity than those unfed parents, the flowers provided for the parents of A. nilaparvate were able to increase the number of parasitoids produced by their progeny. Improving hatchibility, shortening the embryonic developmental time, and increasing the progeny produced by A. nilaparvatae fed with the flowers of T. subulata and C. sulphureus would contribute the overall role of this parasitoid in managing the population of N. lugens in the field.

ACKNOWLEDGEMENT
We would like to thank Ms. Siti Haryati for the discussion on rearing techniques of the parasitoid and Mr. Sudarto for letting us did trapping parasitoids in his rice field. This manuscript is a part of master thesis entitled "Manfaat Bunga   Remark: The parents were fed with flowers and honey but the progeny were not. Mean ± standard deviation followed by the same letter were not significantly different according to LSD (P < 0.05) * Data was transformed to Sin -1 √X.