Metagenomic survey of bacteria associated with the invasive ladybird Harmonia axyridis (Coleoptera: Coccinellidae)

The Asian ladybird Harmonia axyridis is an invasive insect in Europe and the Americas and is a great threat to the environment in invaded areas. The situation is exacerbated by the fact that non native species are resistant to many groups of parasites that attack native insects. However, very little is known about the complex microbial community associated with this insect. This study based on sequencing 16S rRNA genes in extracted metagenomic DNA is the fi rst research on the bacterial fl ora associated with H. axyridis. Lady beetles were collected during hibernation from wind turbines in Poland. A mean ± SD of 114 ± 35 species of bacteria were identifi ed. The dominant phyla of bacteria recorded associated with H. axyridis were Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. Representatives of these phyla are common in the environment, e.g. in the soil, and are often identifi ed as the dominant bacteria associated with arthropods. We also identifi ed animal pathogenic bacteria, such as Burkholderia, Rhodococcus, Chlamydiae and Anaplasmataceae spp. (Neorickettsia helminthoeca and Ehrlichia ovina). We also identifi ed Wolbachia pipientis in a single beetle. This bacterium is a causative agent of reproductive alterations in arthropods. These results support the enemy release hypothesis in the case of this ladybird invasion. Pathogenic bacteria were recorded in only a few samples. Moreover, male-killing bacteria such as Spiroplasma spp., Wolbachia spp. and Rickettsia spp. were only recorded in single insects so they cannot be responsible for the observed alterations in the sex-ratio of the ladybird population studied.


INTRODUCTION
Harmonia axyridis is an important invasive insect (Brown et al., 2008).This ladybird was intentionally introduced into Europe because it is an effective aphid predator (Koch, 2003).However, following its introduction, it has spread rapidly and has adversely affected native coccinellids and other organisms (Tedders & Schaefer, 1994;Koch, 2003;Pervez & Omkar, 2006;Pell et al., 2008;Brown et al., 2011).In Poland, this species was found for the fi rst time in 2007 (Przewoźny et al., 2007) and it is now common throughout this country (Kubisz, 2014).H. axyridis hibernates frequently in anthropogenic structures e.g.houses, wind turbines (Labrie et al., 2008;Raak-Van den Berg et al., 2012;Dudek et al., 2015) in which its overwintering survival is high.It is possible that the invasive success of H. axyridis was enhanced by low parasitism, by which according to the enemy release hypothesis (Roy et al., 2011) an alien species leaves behind its enemies and pathogens in its original territory.Many studies has shown for multiplexing.This reaction contained 5 μl of product from the fi rst PCR reaction, 5 μl of P5 and P7 indices (Nextera Index v2 Kit, Illumina), 25 μl of 2 × KAPA HiFi HotStart ReadyMix kit (KAPA BIOSYSTEMS, USA) and 10 μl of nuclease-free water.Cycling conditions were similar to the fi rst PCR amplifi cation but with the number of cycles reduced to 8.
After each PCR reaction the amplifi ed fragments with tags and adapters were purifi ed using AMPure XP beads (Beckman Coulter Genomic, CA, USA).Amplicon concentrations were quantifi ed using a Qubit fl uorometer (Invitrogen, Carlsbad, CA, USA), normalized to 4 nM and pooled prior to sequencing.To control the purity of DNA libraries, sterile water was used.
The 10 pM library containing 15 pooled indexed samples with 26% spike-in PhiX control DNA was loaded onto the MiSeq sequencing platform.2 × 300 pair end sequencing was performed using the MiSeq Reagent Kit × 3 (600 cycles).
MiSeq Reporter software was used for the secondary data analysis.An average number of 18,800 reads (pass fi lter) per sample was obtained.Microbiome classifi cation was performed based on the Greengenes database (http://greengenes.lbl.gov/).

Statistical analyses
Statistical analyses were done using SPSS v.21 software.

RESULTS
A total of 293,010 pairs of reads were generated for the ladybird microbiome of which 282,402 (96.4%) passed quality fi ltering.The mean number of reads per sample was 19,534 (minimum 1,743 and maximum 63,926).There was a signifi cant infl uence of the method of isolating DNA on the number of the reads (Mann-Whitney U test Z = -3.062;p < 0.001) but not on the quality (p > 0.05).In the ladybirds studied a mean ± SD of 114 ± 35 species of bacteria were identifi ed.The number of pathogens identifi ed strongly depended on the method used to isolate DNA.A mean of 145 ± 50 species of bacteria were identifi ed using kit I and only 50 ± 5 when kit II was used (Mann-Whitney U test Z = -2.819;p = 0.003; Table 1).The most common phyla identifi ed in ladybirds were: Actinobacteria (identifi ed in 15 beetles), Proteobacteria (15), Firmicutes (15), Bacteroidetes (14), Thermotogae (9) and Cyanobacteria (8).Only two species were identifi ed in all the insects sampled: Rhodococcus baikonurensis and R. qingshengii.Aminobacter aminovorans was also dominant and identifi ed in 11 samples.All bacterial DNA identifi ed (from the top seven reads per sample) is presented in Table 2. et al., 2009).Data on bacteria in ladybirds are scarce (Roy et al., 2011).All previous studies were only on a fragment of the bacterial metagenome and focused on specifi c organisms, especially male-killing bacteria, e.g.Spiroplasma spp.(Hurst et al., 1999a;Majerus et al., 1999;Nakamura et al., 2005), Rickettsia spp.(Werren et al., 1994) and Wolbachia spp.(Hurst et al., 1999b).To the best of our knowledge there has, to date, been no studies on the bacterial community of ladybirds.
The main goal of the present study was to describe the metagenomics of the bacterial community associated with H. axyridis in its invaded area.This survey might answer some important questions about pathogen occurrence in ladybirds, e.g. the male-killing bacteria that affect the sex ratios of insects.Moreover, we tested two different DNA isolation kits in order to assess their usefulness for isolating bacterial DNA from insects.

Material collection
The ladybirds used in this study were collected from a wind farm located west of Gołańcz in the Wielkopolska region of Poland (52°57´N, 17°14´E).The wind farm is in an intensive agricultural landscape dominated by oilseed rape and wheat crops.Insects were collected during October and November 2015 from 16 wind turbines (for area description see Dudek et al., 2015).

DNA isolation
DNA from ladybirds was isolated by mechanical lysis (bead beating) using a FastPrep24 instrument and deposits A (MPBiomedicals, CA, USA).Lysis was carried out at a speed of 6.5 m/s for 40 s in 2 cycles (samples were cooled on ice for 5 min between cycles).Further steps in the isolation were performed using a NucleoSpin Tissue Kit (MACHEREY-NAGEL, Germany) and DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers protocols.Samples 1-10 were isolated using the NucleoSpin Tissue kit (Kit I) and 11-15 using the DNA Mini Kit (Kit II).The concentration and purity of isolated DNA were measured spectrophotometrically at wavelengths of A260 and A280 using NanoDrop (ThermoScientifi c, DE, USA).
Cycling conditions were as follows: initial denaturation at 95°C for 3 min; 25 cycles: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s and fi nal extension at 72°C for 5 min.
The second amplifi cation was performed using the PCR product from the fi rst reaction as a template in order to index the samples

DISCUSSION
Sample size was limited by the high cost of this very modern and expensive method; hence the results should be treated with caution.However, because of the novelty of the fi ndings they merit a broad discussion.The dominant phyla of bacteria in the H. axyridis analyzed were Acti-nobacteria, Proteobacteria, Firmicutes and Bacteroidetes.These microorganisms were present in all the insects sampled regardless of the method of isolation used (except for Bacteroidetes, which were not present in one sample).Representatives of these phyla are common in the environment, e.g. in the soil, and are often identifi ed as dominant bacteria associated with arthropods (Carpi et al., 2011;Kaluzhnaya et al., 2012).Thermotogae and Cyanobacteria were also detected in the majority of the samples (9 and 8 respectively).Cyanobacteria are autotrophic organisms existing in almost all environments and often detected associated with other organisms (Whitton, 2012).The detection of Thermotogae bacteria associated with hibernating ladybirds was surprising.These Gram-negative bacteria are extremely thermophilic and occur mostly in thermal springs (Bhandari & Gupta, 2014).Unfortunately, these analyses did not recognize any lower taxonomic level for this phylum.In four ladybirds Tenericutes bacteria were found, to which Spiroplasma spp.belongs; which are common bacteria in the gut and haemolymph of insects and known as male-killing bacteria (Hurst et al., 1999a;Majerus et al., 1999;Nakamura et al., 2005).Interestingly, in one insect, bacteria belonging to the Chlamydiae were detected, which are obligate intracellular pathogens mostly known as the causative agent of sexually transmitted diseases in humans, but also associated with insects (Thao et al., 2003).
We also identifi ed animal pathogenic bacteria, such as Burkholderia, Rhodococcus and Anaplasmataceae spp., such as Neorickettsia helminthoeca and Ehrlichia ovina, which are often transmitted by ticks (Ekner et al., 2011;Matysiak et al., 2016).Another group of mostly endosymbiotic or pathogenic bacteria is the Rickettsiales, which we recorded in four samples.These microorganisms are common in arthropods (Weinert et al., 2009), so it is curious that were recorded them in only a few of the samples of ladybirds studied.The Wolbachia genus also belongs to this family and we identifi ed W. pipientis in a single beetle.This bacterium is the causative agent of reproductive alterations in arthropods (Stouthamer et al., 1999).R. qingshengii is a carbendazim-degrading bacterium recorded in contaminated soil in China (Xu et al., 2007).However, it is also a pathogen of Atlantic salmon (Avendano-Herrera et al., 2011) and recorded in an alpine glacier at a site subject to high levels of human activity (Lee et al., 2011) so this bacterium might be associated with ladybeetles collected from polluted soil around wind turbines.The second species of this genus recorded in all samples was R. baikonurensis, which is a soil bacterium (Yoon et al., 2010) and was isolated for the fi rst time in the air in the Mir space station (Li et al., 2004).R. baikonurensis is known to degrade oil (Lee et al., 2006) and its presence on ladybeetles, like that of R. qingshengii, might be linked with the polluted environment around wind turbines.
This study is the fi rst metagenomic research on the bacterial fl ora associated with the invasive ladybird H. axyridis.Results indicate that the bacterial community associated with H. axyridis consists mostly of symbiotic organisms, widely distributed in the environment.The results also support the enemy release hypothesis applying in the case of this ladybird invasion.Pathogenic bacteria were found in only a few samples.Moreover, male-killing bacteria, such as Spiroplasma spp., Wolbachia spp.and Rickettsia spp., were only found in single insects so they cannot be responsible for the observed alterations in the sex-ratio of the ladybird population studied (the sex-ratio was strongly skewed towards females -65.9% female, chi-square with Yates correction χ 2 = 60.813,p < 0.0001 -unpubl.data).The comparison of the two kits for DNA isolation revealed signifi cant differences (a mean of 145 vs. 50 species of bacteria were detected).The NucleoSpin Tissue Kit isolated much more material than the DNA Mini Kit, which might be a consequence of the greater purity of the DNA extracted by the fi rst kit (Queipo-Ortuno et al., 2008).Thus based on this comparison it is recommended that the Nu-cleoSpin Tissue Kit be used in future research.

Table 1 .
Number of bacteria identifi ed at each taxonomic level using two different methods of isolating DNA.

Table 2 .
Bacteria identifi ed associated with the bodies of ladybirds.For each individual the top seven reads were assigned to a taxonomic status.# -number of ladybird samples where a taxon was identifi ed (15 = 100%).