ESTIMATING TIME DIVERGENCE OF BRACOVIRUS (POLYDNAVIRIDAE) THAT DRIVE THE SPECIATION OF COTESIA SP. (: MICROGASTRINAE)

1AMEYRA AMAN ZUKI, 2MUHAMAD AZMI MOHAMMED, 3BADRUL MUNIR MD. ZAIN, 4SALMAH YAAKOP

1,2,3,4School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia E-mail: [email protected], [email protected], [email protected], [email protected]

Abstract— The specialization of Cotesia species is known to be driven by its endosymbiont Bracovirus. However, the time estimation for this symbiosis happen is still unknown. Studies show that the symbiosis between Bracovirus and its Braconids wasps occured ≈73.7±10MYA, however the time for symbiosis between Cotesia sp. and its bracovirus is unknown. This beneficial parasitoid is very important in biological control program for controlling larva pest that infesting crops. Its endosymbiont helps in the successfulness process of parasitization. In this study, 28S and CrV1 regions were used to study the time estimation of Bracovirus in driving the speciation of Cotesia. In this study, we believed the time divergence for Cotesia speciation occurred is ≈84.05±10MYA and the driving force by bracovirus happens at ≈85.02±10MYA.

Keywords— Molecular clock, speciation, Cotesia, Bracovirus, 28S, CrV1.

I. INTRODUCTION control the population of the Lepidopteran larva from infesting crops in certain area. Cotesia is a genus under subfamily of Microgastrinae Bracovirus was suggested to be evolving from and one of the largest genera in the subfamily. This from 100 MYA [9] and this was cosmopolitan parasitoid wasp genus is really well believed from a single origin [10] and this was known among the biological control experts due to proven that bracovirus only infecting the their function as biological control agent for microgastroid complex in the Braconidae family. The controlling pest in crops [1]. Cotesia will lay their microgastroid complexes are Adeliinae, eggs in the larva of and in many cases , , Khoikhoiinae, the larvae are happen to be the pest for almost all Mendesellinae, Miracinae and Microgastrinae [11]. vegetables and crops all around the world [2]. This virus was believed to symbiosis with Considering the benefits of Cotesia, researchers have Microgastroid complex since ≈73.7±10MYA [12]. been progressing in using this genus for controlling Studies have shown that Bracovirus is helping the the population of the pest, for example Cotesia specialization of their braconids [13]. This plutellae for controlling diamondback moth, Plutella specialization process in the braconids will help the xylostella [3], C. flavipes for controlling stemborer, speciation process of the parasitoid. Hence, this study Chillo partellus [4], C. congregata for controlling was done to estimate the time for bracovirus driving tobacco hornworm [5] and many more. force of Cotesia speciation. The successful of parasitization of Cotesia towards Choosing the right molecular region for the research their host is believed was aided by its own objective is really important in order to achieve the endosymbiont, i.e. bracovirus. Bracovirus is a genus upright results. In this study, two molecular markers from family Polydnaviridae, which are dsDNA virus were used for comprehensively understand the that have been replicated in the ovaries of female symbiosis between parasitoid and its bracovirus. microgastroid [6]. is comprised of two CrV1 marker is used for molecular time divergence of genera, i.e., and Bracovirus. Ichnovirus bracovirus and 28S marker was used to study only infecting parasitoid wasps in the family of molecular time divergence of its parasitoid, Cotesia. Campopleginae (Ichneumonidae), while Bracovirus only infecting wasp in the family of Braconidae [7]. II. MATERIAL AND METHODS Bracovirus is a packaged dsDNA virus that will transfer together with the eggs of the parasitoid into 2.1 Sample collection the larva of Lepidoptera during parasitization. This Parasitoid wasps have been collected using malaise virus will protect the eggs and larva of wasp inside trap from all over Peninsular Malaysia and Cotesia the larva of Lepidoptera from the immune system of species were sorted and kept in the 90% ethanol and the Lepidopteran [8]. Bracovirus will mimic the stored in -20°C. Cotesia species were identified immune system of the Lepidopteran larvae in order to morphologically [14] before proceed to DNA confuse the host therefore protecting the eggs and extraction. The specimen was rinsed under 99% larva of parasitoid until they emerge from pupae of ethanol and distilled water to remove any fungi or Lepidopteran [3]. This mechanism will help to impurities that stick together on the insect’s chitin.

Proceedings of ISER 42nd International Conference, Tokyo, Japan, 11th -12th November 2016, ISBN: 978-93-86291-31-8 23 Estimating Time Divergence of Bracovirus That Drive The Speciation of Cotesia sp. This is to avoid any DNA contamination during 49.7±10MYA. This data is used in the molecular extraction. List of the samples used in this study is clock calibration in the BEAST software. shown in Table 1. III. RESULT AND DISCUSSION Table 1: List of sample used in this study A total of 406 bp and 606 bp of aligned sequences data of 28S and CrV1 region have been successfully obtained to be used in molecular clock analysis. Addition to the sample data, a few sequences from NCBI Genbank were used to aid the analysis and the list of sequences obtained were shown in Table 1. Fig.1 and 2 shows the molecular time divergence of Cotesia using 28S region and bracovirus using CrV1 region. From the 28S and CrV1 cladogram molecular tree, Glyptapanteles indiensis was formed as outgroup as this species is formed at the basal of the tree. The total of species for 28S region and CrV1 region are not parallel with each other because lack of sample obtained from GenBank. However, the total number of the samples for both regions is adequate enough to be use in the analysis. For both trees, C. congregata formed a clade with Malaysia samples Cotesia hallii and the time divergence for this two species are at ≈17.02±10MYA. Generally, the claddings of the species for both trees were quite following their phylogeny classification except for C. flavipes and C. sesamiae, where in 28S tree C. flavipes was formed at the basal of the clade C. chilonis+(C. vestalis+C. sesamiae) with time divergence of ≈26.23±10MYA

while in CrV1 tree, C. flavipes was formed a clade 2.2 DNA extraction, amplification and sequencing with C. sesamiae with time divergence for both Cotesia hallii were dissected in the 1x PBS species are ≈6.37±10MYA. In the CrV1 tree also we (phosphate-buffered saline) to remove the ovaries of can see, C. pluttellae and C. vestalis were formed a the sample and used it in the viral DNA extraction. clade with time divergence of ≈0.59±MYA. As well The DNA extraction has been done using Invisorb® known, taxonomically, these two species are belongs Spin Virus DNA Mini Kit (Stratec Molecular). to the same species, i.e. C. vestalis. Polymerase chain reaction has been done using two pairs of primers, i.e. 28S for Cotesia hallii and CrV1 For time divergence of Cotesia species, this genus for Cotesia hallii bracovirus. The primers used as was formed at ≈84.05±10MYA, while the symbiosis follow; 28S F 5’- AGA GAG AGT TCA AGA GTA happen between this parasitoid and its bracovirus at CGT G-3’ [15], 28SR 5’- TTG GTC CGT GTT TCA ≈85.02±10MYA. This result is quite bizarre because AGA CGG G-3’ [16], CrV1087F 5’-ATG TCA CTC it seems that the symbiosis is happen before the genus GTC AAA AGT GC-3’and CrV2107R 5’- AAA GTT is formed itself. However, the case is vice versa. TGC GAT GGG GTT GT-3’ [17]. The successful Following the studies from Herniou et al. [19], the PCR products were sent to 1stBase company study suggested that the adaptive selection of the (Serdang, Selangor) for sequencing analysis. Bracovirus gene is actually driving the specialization of Cotesia parasitoid wasps. That is means, the 2.3 Molecular Clock analysis speciation and specialization of the parasitoid species Divergence time estimation for both 28S and CrV1 is actually driven by its Bracovirus [13]. This is regions were run using BEAUTi and BEAST [18]. because, Bracovirus have to adapt with the host in The molecular clock analyses were run using order to successfully aiding the parasitization process uncorrelated relaxed clock with 100,000,000 [20]. Going through the adaptation for years, make generation by MCMC approach. To calibrate the the Bracovirus have to select the best gene for clock with the fossil, we refer Whitfield [12] that survival. The selective adaptation of the gene inside suggested the microgastroid lineage carrying the parasitoid wasp leads to speciation of the wasps polydnavirus between 93 and 45 MYA. From that itself. Henceforth, the molecular time divergence for study, Whitfield concluded that symbiosis of Bracovirus gene will be before the speciation of the bracovirus and its braconids happened73.3±10MYA parasitoid happened. and between bracovirus and Microgastrinae is

Proceedings of ISER 42nd International Conference, Tokyo, Japan, 11th -12th November 2016, ISBN: 978-93-86291-31-8 24 Estimating Time Divergence of Bracovirus That Drive The Speciation of Cotesia sp. ACKNOWLEDGEMENTS

This research was funded by the FRGS/1/2014/SG03/UKM/02/1 and GUP-2014-029 research grants.

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