DETECTION OF WOLBACHIA IN LARVAE OF LOXOSTEGE STICTICALIS (PYRALOIDEA: CRAMBIDAE) IN EUROPEAN AND ASIAN PARTS OF RUSSIA

Beet webworm Loxostege sticticalis is a notorious pest widely distributed on the territory of Eurasia. Its outbreaks cause severe damage to crops in Russia and China. Here Wolbachia infection is reported for the first time in L. sticticalis. Larvae were sampled in Rostov, Saratov, Irkutsk Regions and Republic of Buryatia in 2005–2013. Primers targeting the wsp gene were used for the PCR screening of Wolbachia. Among 148 larvae, 35 were Wolbachia positive. Wolbachia prevalence rate ranged from 21 to 40 % in the Asian and from 0 to 47 % in the European part of Russia. The combined sample subsets were compared for European versus Asian part of Russia and 2005–2009 versus 2010–2013 timeframes. The prevalence rates of Wolbachia were not significantly different between two parts of Russia, but the endosymbiont presence (estimated for the total dataset) increased with time within the observation period.


Introduction
Beet webworm Loxostege sticticalis L. is a notorious pest of numerous crops in European and Asian parts of Russia (Frolov et al., 2008), as well as in Northern China (Chen Xiao et al., 2008). Screening insect populations for naturally occurring parasites and pathogens is crucial for understanding the dynamics of pest populations. In particular, obligate intracellular parasites, such as Microsporidia, play an essential role in L. sticticalis density dynamics (Frolov et al., 2008). So far, other intracellular symbionts in populations of L. sticticalis have not been reported.
Bacteria of the Wolbachia genus are widespread endosymbionts of arthropods (Jeyprakash, Hoy, 2000). In certain species of Lepidoptera, Wolbachia may contribute to population biology of the hosts (Salunkhe et al., 2014), as it regulates reproductive processes (including sex determination) and influences host vitality and fertility in direct or indirect ways (Kageyama et al., 2002;Kageyama, Traut, 2004). The knowledge of Wolbachia distribution in insect populations is therefore of great interest, being important for a better understanding of the mechanisms underlying regulation of pest density dynamics (Sumi et al., 2017). Here, we report the first results of screening Wolbachia infection in L. sticticalis populations.

Materials and Methods
Beet webworm larvae were collected on crops and weeds in European and Asian parts of Russia (Fig. 1A). Insects were fixed with ethanol and stored at -20 °C. Total DNA was extracted using a simplified protocol of Sambrook et al. (1989) without addition of phenol. For quality control of DNA samples, the primers LepF1/LepR1 (Hebert et al., 2004) specific for the barcoding region of mitochondrial cytochrome oxidase subunit I (COI) were used. DNA samples producing a specific signal with COI-targeted primers were selected for further analysis. The Wolbachia infection was detected by amplification with primer set wsp81F/wsp691 (Zhou et al., 1998), specific to the locus of Wolbachia surface protein (wsp). We used DreamTaq Green PCR Master Mix (Thermo Fisher Scientific) with the following cycling conditions: initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 1 min, annealing at 54 °C for 1 min, elongation at 72 °C for 1 min, and final elongation step of 72 °C for 5 min. The amplicons were visualized using electrophoresis in 1 % agarose gels with GeneRuler Ladder Mix molecular weight marker, 75-20000 bp (Thermo Fisher Scientific). The 95 % confidence intervals were estimated using the Clopper-Pearson method (Clopper, Pearson, 1934) which is routinely used when Wolbachia prevalence rates in small samples are examined (Yudina et al., 2016, Bykov et al., 2019. Estimates of the data reliability were obtained using the exact Fisher's test (Fisher, 1922), Pearson's chi-square criterion and chi-square criterion with Yates correction (Yates, 1934).

Results and Discussion
PCR with primers specific for wsp gene fragment of Wolbachia has yielded amplicons with the expected size of ~600 bp (Fig. 1B)   To further test possible differences in Wolbachia prevalence rates over time and place, we have compared the combined sample subsets of European versus Asian part of Russia and 2005Russia and -2009Russia and versus 2010Russia and -2013 timeframes. In European part of Russia (Salsk + Saratov), the average Wolbachia prevalence rate have been 19.0 % (N=84), while in Asian part (Irkutsk + Kabansk) this index has reached 29.7 % (N=64). According to Pearson's chi-square criterion (χ 2 =2.271), the prevalence rates of Wolbachia does not depend on the geographical origin of the sampled populations, which indirectly confirms the conclusion that the beet webworm populations belong to a single metapopulation (Jiang et at., 2010) Prevalence rates of Wolbachia in insect hosts may significantly vary over time and space. For example, in pyraloid moths of the genus Ostrinia, Wolbachia has been found in all examined populations in European part of Russia, and the prevalence rates have depended on the species and the forage plant (Tokarev et al., 2018). Long-distance migrations are likely to provide symbiont exchange between local populations of the beet webworm, but various factors may affect the temporal dynamics of Wolbachia infection, revealed in the present study.
According to the Russian Agricultural Center (https:// rosselhoscenter.com/), low density of beet webworm was reported in the Russian Federation in 2005and 2006. From 2008to 2014, the period of relatively high density was observed in Russia, with maximum in 2009, when the pest outbreaks occurred throughout the entire pest area from the Southern and Central Federal Districts to the Far Eastern Federal District. We have noticed a trend that during the period of low pest abundance, infection rate of Wolbachia was at its minimum, while during the period of high abundance, it was increasing over time. A long depression period of the pest, observed from 2015 to 2018, has been followed by an increase in the number of the beet webworm in Siberia. In the coming years, we expect to collect more data and to verify the relationship between the pest number and the frequency of Wolbachia infection.
The Wolbachia infection has been found for the first time in populations of L. sticticalis in the present study. Although the sampling sites are not numerous, it is obvious that the bacterium is present in the majority of local samplings. The beet webworm tends to form a single metapopulation on the territory of Eurasia due to its high migratory activity. In a given locality, the endosymbiotic bacterium may change its state over time from absence (presence at undetectable levels) to presence in a half of the insect population, as shown for the samplings from Saratov. The examined dataset does not allow to determine whether the fluctuations of Wolbachia prevalence rates is adaptive or stochastic. Further studies are necessary to elucidate the genetic diversity of Wolbachia and its biological role in populations of the beet webworm.