Wolbachia endosymbiont infection in two Indian butterflies and female-biased sex ratio in the Red Pierrot, Talicada nyseus 1 2 1, KUNAL ANKOLA , DOROTHEA BRUECKNER and HP PUTTARAJU * 1Division of Biological Sciences, School of Natural Sciences, Bangalore University, Bangalore, India 2Department of Biology, University of Bremen, Bremen, Germany *Corresponding author (Email, [email protected]) The maternally inherited obligate bacteria Wolbachia is known to infect various lepidopteran insects. However, so far only a few butterfly species harbouring this bacterium have been thoroughly studied. The current study aims to identify the infection status of these bacteria in some of the commonly found butterfly species in India. A total of nine butterfly species belonging to four different families were screened using PCR with Wolbachia-specific wsp and ftsZ primers. The presence of the Wolbachia super group ‘B’ in the butterflies Red Pierrot, Talicada nyseus (Guerin) (Lepidoptera: Lycaenidae) and Blue Mormon, Papilio polymnestor Cramer (Papilionidae), is documented for the first time in India. The study also gives an account on the lifetime fecundity and female-biased sex ratio in T. nyseus, suggesting a putative role for Wolbachia in the observed female-biased sex ratio distortion. [Ankola K, Brueckner D and Puttaraju HP 2011 Wolbachia endosymbiont infection in two Indian butterflies and female-biased sex ratio in the Red Pierrot, Talicada nyseus. J. Biosci. 36 845–850] DOI:10.1007/s12038-011-9149-3 1. Introduction infected by Wolbachia. It has been shown that the presence of particular clades of Wolbachia cause feminization and The maternally inherited endosymbiotic α–proteobacteria cytoplasmic incompatibility in the common grass yellow called Wolbachia is known to infect 15%–75% of insect butterfly, Eurema hecabe (Hiroki et al. 2004). However, species (Werren and Windsor 2000; Jeyaprakash and Hoy recent studies also suggest that some E. hecabe infected with 2000; Puttaraju and Madhu 2002; Puttaraju and Prakash a single strain of Wolbachia have two distinct reproductive 2005a, b; Prakash and Puttaraju 2007). This endosymbiont phenotypes (Narita et al. 2007). Two male-killing bacteria is known to cause some reproductive abnormalities in its insect were reported in Acraea encedon, which were shown to hosts, such as cytoplasmic incompatibility (Laven 1967; belong to the same strain (Jiggins et al. 2001), and this Breeuwer and Werren 1990; Breeuwer et al. 1992;O’Neill particular strain is also known to cause male killing in et al. 1992; Prakash and Puttaraju 2007), parthenogenesis Hypolimnas bolina, indicating that Wolbachia might have (Stouthamer et al. 1990, 1993; Arakaki et al. 2001), male moved between host species, retaining its phenotype killing (Fialho and Stevens 1997, 2000;Hurstet al. 1999, (Dyson et al. 2002). Further, the male-killing Wolbachia 2000) and feminizing of genetic males (Rousset et al. 1992; has been reported in the Indo-Pacific populations of Hiroki et al. 2002). Wolbachia exist in 11 clades (A to K) Hypolimnas bolina, which causes sex ratio distortion referred as super groups, which infect not only wide range of compared to the normal sex ratio (Charlat et al. 2005). It insects but also many non-insect invertebrates. However, the is also known that apart from Wolbachia,someother super groups A and B are known to infect the insect kingdom bacteria like Spiroplasma can cause male killing in (Werren et al. 1995; Prakash and Puttaraju 2007). The butterflies by killing the male embryo (O’Neill et al. association of Wolbachia with order Lepidoptera is very well 1997; Hurst and Jiggins 2000; Jiggins et al. 2000; Tabata studied, but only a few species of butterflies are known to be et al. 2011). Keywords. Papilio polymnestor;PCR;sexratio;Talicada nyseus; Wolbachia http://www.ias.ac.in/jbiosci J. Biosci. 36(5), December 2011, 845–850, * Indian Academy of Sciences 845 846 K Ankola, D Brueckner and HP Puttaraju The Red Pierrot, Talicada nyseus (Guerin) (Lepidoptera: laboratory carefully in butterfly collecting cages, identified Lycaenidae), is a small butterfly found in many parts of and separated with respect to their sex. India and Sri Lanka (Karunaratne et al. 2002). It is commonly found around semi-arid plains, degraded patches 2.2 Rearing of Talicada nyseus of evergreen forests and semi-evergreen forest, gardens, hill stations and forests. It is abundantly found near its food The adult T. nyseus collected were released in one cage with plant Kalanchoe spp. (Saxifragales: Crassulaceae). In India nectar and host plants for mating and increase of population. this butterfly is widely distributed in the peninsular area and The other developmental stages such as eggs, larvae and is also known to colonize the foothills of the Himalayas due pupae were reared in aerated boxes until they metamor- to change in habitat (Singh 2005). The larvae of this species phosed into adult. The emerged adults were released in the are known to feed on the mesophyll tissues of the leaves of its same cages for increasing the stock population. host plant Kalanchoe spp. without disturbing the upper and lower epidermis. An adult butterfly usually feeds on nectar 2.3 Experimental setup but is also known to feed on lichens (Karunaratne et al. 2002). It is further estimated that T. nyseus can damage most One virgin female along with two males from the stock of its host plant species that are used for ornamental purpose. population were released separately in a cage along with During a regular field survey in Bannerghatta Biological host and nectar plants for mating and egg laying. For nectar Park, Bangalore, it was found that the population of Lantena plants were used. A total of 12 individual females T. nyseus was dwindling and males were relatively rare. (n=12) were used for the study in each cage separately. The Reliable data on the fecundity and sex ratio in T. nyseus are fecundity was estimated manually by repeatedly counting not available and this study gives an account of female- the number of eggs on the host plants at regular time biased sex ratio in this butterfly species. This kind of sex intervals (twice a day until the female died). The egg-bearing ratio distortion or female-biased sex ratio has been previ- plants were carefully taken out of the cages and kept separately ously reported in some other butterflies from different under observation. The hatchability was ascertained by families. This female-biased sex ratio is known to be caused counting hatched eggs on host plants. The larvae were grown by Wolbachia in some butterfly species. In Acraea encedon, to complete metamorphosis on Kalanchoe leaves. The pupae female-biased sex ratio was first reported by Poulton in were counted and taken out from the plants and reared 1914 and it was thought that this sex ratio distortion was the in aerated boxes. The adults emerged were then result of meiotic drive. Later it was proved that the cause for examined morphologically to determine their sex ratio. sex ratio distortion was the presence of the maternally inherited bacteria Wolbachia (Jiggins et al. 1998, 1999). 2.4 Molecular detection of Wolbachia infection Eurema hecabe is known to be infected by different types of Wolbachia strains (Hiroki et al. 2002, 2004; Narita et al. The genomic DNA was isolated by two methods viz the 2007) and shows various types of reproductive anomalies phenol-chloroform isoamyl alcohol method (Sambrook such as cytoplasmic incompatibility, feminization and et al. 1989) and the Kit method (Ultraclean Tissue and male killing. Cells DNA isolation Kit by MO BIO Laboratories, Inc.). The present study was carried out to identify the infection The DNA from nine species of butterflies belonging to status of Wolbachia and its super group in some of the four different families was isolated by the phenol- butterfly hosts that are commonly found in India. Further, chloroform isoamyl alcohol method for initial screening the present study also provides information on lifetime of Wolbachia infection, whereas the DNA from the female fecundity and sex ratio of T. nyseus harbouring Wolbachia T. nyseus used for the experiment was isolated by the Kit infection under controlled environmental conditions. method. The extracted DNA was quantified by agarose gel (0.8%) electrophoresis. 2. Materials and Methods Molecular diagnosis of Wolbachia and its super groups A and B were done by Wolbachia-specific PCR assay using 2.1 Collection of butterflies specific primers like wsp 81f 5′-TGG TCC AAT AAG TGA TGA AGA AAC-3′, wsp 691r 5′-AAA AAT TAA ACG The butterfly species were collected from their natural CTA CTC CA-3′ (Dyson et al. 2002), ftsZ Adf 5′-CTC habitats in the forest areas of Bannerghatta Biological Park, AAG CAC TAG AAA AGT CG-3′ ftsZ Adr 5′-TTA GCT Bangalore, and Jnana Bharathi Campus, Bangalore University. CCT TCG CTT ACC TG-3′ (Prakash and Puttaraju 2007) Butterflies were collected using a butterfly collecting net. The and ftsZ Bf 5′- CCG ATG CTC AAG CGT TAG AG-3′ ftsZ collection was done in the morning hours from 8.30 a.m. to Br 5′-CCA CTT AAC TCT TTC GTT TG-3′ (Prakash and 1.00 p.m. The collected species were transferred to the Puttaraju 2007). PCR amplification was carried out with J. Biosci. 36(5), December 2011 Wolbachia endosymbiont in Red Pierrot 847 Table 1. The PCR results showing the infection status of Wolbachia super groups in butterfly species belonging to four different families Family Butterfly species Number of individuals Number of individuals infected Super group status screened with Wolbachia Nymphalidae Ariadne merione Danaus genutia Tirumala limniace 30 None – Danaus chrysippus Euploea core Papilionidae Papilio demoleus 30 None – Papilio polymnestor 30 30 B Lycaenidae Talicada nyseus 30 30 B Pieridae Catopsilia pyranthe 30 None – A total of 30 individuals from each species collected from the study area were screened for their Wolbachia infection.