Prevalence of Endosymbionts in Polish Populations of Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

Colorado potato beetle (CPB, Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) is one of the most serious insect pest feeding on wild and cultivated Solanaceae plants. This pest poses a significant threat to potato crops. CPB originated from North America but has become widespread and has adapted in new localizations. Currently, it is reported in many countries worldwide. Endosymbiotic bacteria might have an influence on insect adaptation to new conditions. They are known to play a role in invasiveness of insect hosts and to facilitate colonization of new niches; however, information on endosymbionts of the CPB is very limited. In this study, we screened CPB populations collected from 20 evenly distributed locations in Poland for the presence of Arsenophonus, Cardinium, Wolbachia, and Flavobacterium. We found the presence of Flavobacterium in the studied insects. Little is known about CPB-endosymbionts interactions, thus this study may provide a reference for future studies in this subject.

The Colorado potato beetle [CPB, Leptinotarsa decemlineata (Say 1824), Coleoptera: Chrysomelidae] is an important insect pest of potato crops worldwide. It originates from North America where it first developed on wild Solanaceae plants and then adapted to cultivated potatoes (Alyokhin et al). In Poland, the average number of plants exhibiting the feeding symptoms of L. decemlineata is 23.3% of the crops. This number varies between different regions of Poland, from 5% up to 100% (Walczak et al. 2010). The minimal number of larvae considered to cause economically significant crop losses in Poland is 60 larvae on one potato plant (Pawińska 2003) and two adult individuals per 25 plants (Erlichowski 2009). It was reported that CPB’s destruction of leaf surface, by above 15%, means up to a 28% crop loss from one hectare, which means around 7 tons of potato crops (Kowalska 2010).

One of important biological factors influencing occurrence and viability of the insect is symbiotic bacteria. Bacterial endosymbionts are obligate intracellular, Gram-negative bacteria that may belong to alpha-Proteobacteria (Anderson and Karr 2001, Bowman 2011), beta-Proteobacteria (Kono et al. 2008), gamma-Proteobacteria (Novakova et al. 2009), Bacteroidetes, and Mollicutes (Majerus 2006). They are cyptoplasmically inherited and tend to be concentrated in the reproductive tissues of insects (Werren 1997). Different endosymbionts can co-inhabit with each other inside the bacteriocytes and are vertically transmitted via the eggs (Skaljac et al. 2010).

Number of reports describes an influence of endosymbionts on various aspects of insects biology like the sensitivity to heat stress and other environmental factors (Montllor et al. 2002), defense against natural enemies like predators or parasites (Oliver et al. 2005, 2010), nutrition (Koga et al. 2003, Brownlie et al. 2009), genetic differentiation (Charlat et al. 2009), and reproduction (White et al. 2009). Endosymbionts may also cause a cytoplasmic incompatibility between insects infected with endosymbiont and not infected (Brownlie et al. 2009). They can also influence plant–insect interactions to benefit of their insect host. One of the examples is the case of the phytophagous leaf-mining moth Phyllonorycter blancardella (Lepidoptera), which relies on bacterial endosymbionts to manipulate the physiology of its host plant. The result is a production of photosynthetically active green areas in the senescent leaves (Kaiser et al. 2010). Another example is the report concerning Wolbachia, an endosymbiont of the root herbivore Diabrotica virgifera, that may suppress the induction of transcripts associated with defense response in maize roots (Barr et al. 2010). This would explain why plants that are attacked once become more vulnerable to subsequent D. virgifera attacks. In another experiment (Robert 2013), D. virgifera consistently cured from Wolbachia and subsequently put on maize leaves did not change the level of the induction of defense marker genes. Kikuchi et al. (2012) reported that infection with an insecticide-degrading bacterial symbiont immediately establishes insecticide resistance in pest insects. The experiments performed on bean bug Riptortus pedestris and allied stinkbugs showed that those insects harbor mutualistic gut symbiotic bacteria of the genus Burkholderia, which are acquired by nymphal insects from environmental soil every generation.

Since each insect species has its own set of endosymbiont species (Thao and Baumann 2004), information about the endosymbionts population in one insect species cannot directly refer to another investigated insect species. There is limited information available on the endosymbiont range in populations of L. decemlineata. CPB is a very important pest affecting potato plants throughout the world. Limiting the population of the CPB is a constant challenge, and therefore, there is a need to know more about its biology. In this study, we have undertaken to investigate for the first time the presence and the frequency of endosymbiont infections in populations of CPB collected from potato fields in Poland—one of the biggest potato growing countries in Europe. Our finding might give a preliminary insight into endosymbiotic infections of CPB and open the possibility for the further study on the CPB–bacteria interactions as well as their impact on the insect–plant interactions.

Materials and Methods

Collection of Specimens

Twenty populations of CPB collected in 2011 from various, evenly distributed, locations in Poland were investigated. The list of locations, their GPS coordinates, and their graphic representation are shown in Fig. 1.

Fig. 1.

An occurrence of endosymbiotic bacteria in L. decemlineata populations collected from various localizations in Poland. The black circle next to Arabic number means a place of collection of sample. The Roman numbers refer to climatic zones of Poland. A name of the genus next to a collection site means the location where the given endosymbiont was detected. NW, north west; NE, north east; SW, south west; SE, south east. List of sampling locations and their GPS parameters: 1-N51 28.001 E14 57.536; 2-N52 30.242 E15 01.138; 3-N53 25.923 E15 04.730; 4-N50 28.391 E17 00.796; 5-N51 56.786 E17 03.003; 6-N52 55.433 E16 57.701; 7-N54 30.597 E16 57.902; 8-N50 01.742 E19 04.143; 9-N51 08.852 E19 08.203; 10-N52 41.329 E18 54.122; 11-N53 59.112 E19 00.039; 12-N49 29.290 E20 59.105; 13-N50 29.975 E21 04.211; 14-N51 54.874 E20 56.375; 15-N53 29.051 E20 58.015; 16-N49 57.863 E22 53.821; 17-N50 58.104 E23 01.627; 18-N52 02.008 E23 00.207; 19-N53 01.442 E23 00.410; 20-N53 59.956 E23 04.128.

All insect samples were collected from potato fields. From each location, five (or six for one location) randomly harvested specimens were collected, including both imagos and larvae. Collected specimens were fixed in the field in 96% ethanol and stored at –20°C until analysis. In total, 101 randomly collected individuals of CPB from 20 locations in Poland were tested against the presence of the endosymbionts. Among them, there was 21 males (M), 20 females (F), and 60 larvae (L).

Endosymbiont Screening

For each location, the sex of the collected adults was determined under the binocular. For further investigation, the genomic DNA of male, female, and larva of L. decemlineata was isolated. The DNA was extracted separately for males, females, and larva in each population. Each location was represented in further investigation. The haplotype of collected CPB individuals was determined in the previous study on the basis of mtCOI and mtCOII gene sequencing (Jeszke et al. 2014).

DNA Isolation and Polymerase Chain Reaction

The genomic DNA of collected insects was isolated using the NucleoSpin Tissue Kit (Macherey-Nagel, Germany). Purified genomic DNA was dissolved in 100 ml of TE buffer (pH 8.0) and stored at −20°C for further analysis. All genomic DNA samples were screened for the presence of endosymbiotic bacteria from following genera: Arsenophonus, Cardinium, Flavobacterium, and Wolbachia. The presence of endosymbiotic bacteria in each individual was determined by polymerase chain reaction (PCR)-gel analyses of 16S rRNA (Cardinium), 23S rRNA (Arsenophonus and Flavobacterium), or wsp gene (Wolbachia) using genus-specific PCR primers (Pan et al. 2012). As a positive PCR control, we used Arsenophonus (GenBank accession number: KR709153) and Flavobacterium (KT122797, this study).

For screening purposes, the PCR amplifications were performed in 10 µl volume reactions containing PCR buffer (1X) (Life Technologies, Carlsbad, CA), MgCl2 (2.5 mM), dNTP (0.2 mM), forward primer (0.4 µM/µl), reverse primer (0.4 µM/µl), Taq polymerase (0.1 U/µl) (Life Technologies, Carlsbad, CA), and DNA template (100–150 ng//µl). For PCR cycling, the touch down PCR protocol was used (Korbie and Mattick 2008). The resultant PCR products were electrophoresed on a 1.0% agarose gel in a 0.5 x SB buffer and visualized in UV light using Midori Green (Nippon Genetics, Dueren, Germany) as a fluorescent dye. The identities of the expected bands for randomly selected symbionts from each genus were verified by subsequent sequencing of PCR products. DNA sequences of the sequenced PCR bands were submitted to the GenBank database (Table 1). Additionally, a dendrogram of the 23S rRNA partial gene sequences from obtained PCR products and analogical sequences of other tested endosymbionts derived from the GenBank was constructed. The tested isolates are F1L, F2L, F3L, F6M, F12M, F13M, and F18M and were marked with a black square in Fig. 2. The multiple sequence alignments were performed using the ClustalW, and phylogenetic analysis was carried out by the Neighbor-Joining algorithm implemented with MEGA6. Bootstrap values for phylogenetic comparisons were based on 1,000 replicates.

Table 1.

Prevalence and infection rates of endosymbionts of L. decemlineata

Endosymbiont genus	Detected infection rate in males (%)	Detected infection rate in females (%)	Detected infection rate in larvae (%)	Total detected endosymbiont infection rate (%)	Prevalence
Arsenophonus	0	0	0	0	—
Cardinium	0	0	0	0	—
Flavobacterium	23.8 (5 out of 21)	0.2 (1 out of 20)	10 (6 out of 60)	11.88 (12 out of 101)	1, 2, 3, 6, 10, 12, 13, 15, 17, 18, 20
Hamiltonella	0	0	0	0	—

The numbers describe the geographic localization where CPB were collected and refer to the Figure 1 where all localizations were marked.

Fig. 2.

Dendrogram constructed by neighbor-joining analysis of the 23S rRNA partial sequences gene from obtained PCR products and analogical sequences of other tested endosymbionts derived from the GenBank. The obtained isolates are marked as F1L, F2L, F3L, F6M, F12M, F13M, and F18M. The nucleotide sequences were analyzed using the BioEdit and MEGA6 software. The multiple sequence alignments were performed using the ClustalW program. Phylogenetic analysis was carried out by the neighbor-joining algorithm implemented with MEGA 6. Bootstrap values for phylogenetic comparisons were based on 1,000 pseudoreplicates.

Endosymbionts Infection Rate and Statistical Analysis

The total number of tested individuals was recorded to calculate their infection frequencies according to this formula: the number of individuals infected by a given symbiont/the total number of individuals screened (Table 1). The values obtained in each replicate were statistically analyzed. The impact of sex and life stage on the infection frequencies of the symbionts was calculated. For comparison of several dependent groups in dichotomous scale, a Cochran Q test was used. For verification of significant relationship between variables, we used independence Chi-square test. For all analyses, the level of significance was α = 0.05, and the P value was then calculated. Statistical analysis was performed using STATISTICA v.12 (StatSoft Poland).

To investigate whether there is a relationship between the geographic localization of L. decemlineata populations and their endosymbionts infection rate, we proposed the division of Poland into climatic zones, described in the European and Polish standard: PN-EN 12831. According to this division, Poland is divided into five climatic zones marked with Roman numerals. Zone numbers are from I to V. Each climatic zone has its own characteristic average annual temperature: I: 7.7°C, II: 7.9°C, III: 7.6°C, IV: 6.9°C, and V: 5.5°C.

Results

A Country-Wide Screening for Endosymbiotic Bacteria in L. decemlineata

A country-wide survey of endosymbiont contents in L. decemlineata populations collected from potato plantings was done. All collected populations possessed mitochondrial haplotype 1 (on the basis of mtCOI and mtCOII sequences [Jeszke et al. 2014]), which is prevalent in Europe (Grapputo et al. 2005). In collected individuals, we detected the presence of only one endosymbiont genus: Flavobacterium in 11 following locations: 1, 2, 3, 6, 10, 12, 13, 15, 17, 18, and 20 (Table 2). The obtained randomly selected PCR products sequences comparison with analogical Flavobacterium sequences derived from the GenBank is presented in Table 3.

Table 2.

Endosymbionts screening results

Location	Arsenophonus			Cardinium			Flavobacterium			Wolbachia			Sequenced DNA template
	M	F	L	M	F	L	M	F	L	M	F	L
1	−	−	−	−	−	−	−	−	+	−	−	−	F1L
2	−	−	−	−	−	−	−	−	+	−	−	−	F2L
3	−	−	−	−	−	−	−	−	+	−	−	−	F3L
4	−	−	−	−	−	−	−	−	−	−	−	−
5	−	−	−	−	−	−	−	−	−	−	−	−
6	−	−	−	−	−	−	+	−	−	−	−	−	F6M
7	−	−	−	−	−	−	−	−	−	−	−	−
8	−	−	−	−	−	−	−	−	−	−−	−	−
9	−	−	−	−	−	−	−	−−	−	−	−	−
10	−	−	−	−	−	−	−	−	+	−	−	−	F10L
11	−	−	−	−	−	−	−	−	−	−	−	−
12	−	−	−	−	−	−	+	−	+	−	−	−	F12M
13	−	−	−	−	−	−	+	−	−	−	−	−
14	−	−	−	−	−	−	−	−	−	−	−	−
15	−	−	−	−	−	−	−	−	−	−	−	−
16	−	−	−	−	−	−−	−	−	−	−	−	−
17	−	−	−	−	−	−	−	−	−	−	−	−
18	−	−	−	−	−	−	+	−	−	−	−	−	F18M
19	−	−	−	−	−	−	−	−	−	−	−	−
20	−	−	−	−	−	−	−	+	−	−	−	−

M, L. decemlineata male; F, female; L, larva; +, presence of the expected size of PCR product; −, absence of PCR product.

Table 3.

BLAST sequence similarity (%) and sequence coverage (%) of the 23S rDNA sequences of the tested isolates in comparison with analogical Flavobacterium sequences derived from the GenBank

Nb.	Tested strain	GenBank accession number (this study)	Sequence length of the tested strain (bp)	Query coverage (%)	Identity (%)	Flavobacterium GenBank accession number
1.	F1L	KT122797	552	100	94	NR076656
						CP001673
2.	F2L	KT122798	477	96	92	NR076656
						CP001673
3.	F3L	KT122799	307	100	93	NR076656
						CP001673
4.	F6M	KT122800	534	100	87	NR076656
						CP001673
5.	F12M	KT122801	529	100	86	NR076656
						CP001673
6.	F13M	KT122802	438	100	93	NR076656
						CP001673
7.	F18M	KT122803	517	100	92	NR076656
						CP001673

Endosymbionts Infection Rate

The insight into results showed that Flavobacterium symbionts were mostly detected in larva or males of CPB (in localization 12, it was detected in both larva and male. Endosymbiont in insect females was detected only in one location (20).

Within the tested sample (101 insect individuals), we evaluated the association of the specific endosymbiotic bacteria infection with the sex and life stage of the CPB. Out of 101 tested specimens, 11.88% was infected with Flavobacterium. Among males tested, the infection was 23.8%, among females: 5%, and among larvae: 10% (Table 2). The P value that we obtained in performed Cochran Q test equals P = 0.014996 for α = 0.05, which allows us to conclude that the sex and stage of development of insects has the influence on their susceptibility to Flavobacterium infection. The results of performed Pearson Chi-square test (P = 0.13798) shows that there is no correlation between the dynamics progress of Flavobacterium infection between groups of males, females, and larvae.

Flavobacterium presence was detected in all five tested climatic zones of Poland (Table 1, Fig. 1), which refers to the average annual temperatures between 5.5°C and 7.9°C. No presence of infection by Arsenophonus, Cardinium, and Wolbachia was detected regardless of the collection sites, sex, or life stages of the L. decemlineata. Also because of the small amount of data on the occurrence of bacteria on five analyzed areas (n < 20), the statistical analysis cannot be performed.

Discussion

The CPB is an extremely well-adapted species living on its host plants which mainly belong to the Solanaceae. It spread quickly from North America to Asia and continental Europe where it constitutes a serious threat to potato and eggplant cultivations. The research on the CPB’s genetic diversity led researchers to the conclusion that the presence of CPB in Europe resulted from a single successful founder event (Grapputo et al. 2005). In spite of the significance of this harmful pest for Solanaceae as well as its prevalence, there is still a lot to learn about the biology, genetics, and the endosymbiotic content of the CPB.

Numerous invertebrates harbor endosymbiotic microorganisms inside special structures called bacteriomes, in the abdomen of the host insect (Ishii et al. 2013). Every insect species has its own set of endosymbiotic species that can vary greatly, even within the same insect species. The variation depends on factors such as sex, life stage, biotype, and geographic location of the host plant (Duron et al. 2008, Kaiser et al. 2010, Pan et al. 2012).

Performed survey was done on insects collected across the Poland and might constitute the introduction for further detailed analysis of insect endosymbiotic infections worldwide, both describing the content and frequency of CPB-associated bacteria, which might vary depending on the location and conditions from which given CPB populations were collected. Although the described report concerns the single country, and the sample size is not high, the trapping locations are distributed evenly throughout Poland (average distance equals 149.5 km) and represent various climatic and geographic conditions in the country being the one of the biggest potato producers in the world.

In this study, we have undertaken to screen for the presence of various genera of endosymbiotic bacteria. In this report, we found that Polish population of CPB has been infected by Flavobacterium symbionts. This result differs with the outcomes of the other experiment, where 136 insect species were tested against the presence of endosymbiotic bacteria, and as the result, six genera were pointed out as the most frequently present in the arthropods: Wolbachia, Portiera, Hamiltonella, Rickettsia, Cardinium, and Arsenophonus (Duron et al. 2008). The same symbiotic bacteria were investigated in several other studies, in various insect hosts, among others in Bemisia tabacii (Pan et al. 2012), Phyllonorycter blancardella (Kaiser et al. 2010), or ladybird beetle (Coccinella spp.). The conclusion in all mentioned papers is that the level of infection rate and endosymbiont species composition for each insect species is variable and can be different even for the same insect species collected from various locations. There can be also essential differences in the endosymbiotic content of the insects (Duron et al. 2008, Kaiser et al. 2010, Pan et al. 2012). In this study, we did not find the presence of Arsenophonus, Cardinium, and Wolbachia in the tested CPB populations.

Next, we also tried to estimate preliminarily the impact of several factors like sex, life stage, and geographic location of the insect, on the infection frequency of endosymbiotic bacteria in L. decemlineata. Our results show that out of all tested male insect individuals, 23.8% were infected with Flavobacterium (Table 2). The infection rate of Flavobacterium for all tested female insect individuals was much lower (0.2%) and for larvae it was 10% (Table 2). In previously mentioned studies, the endosymbiotic infection rates for other tested insects were significantly different for each sex, like in the case of a Hamiltonella where its prevalence was 88.9% for males and 12.5% for females (Pan et al. 2012), or without difference, like in the case of P. blancardella where Wolbachia was the only endosymbiont reported and displayed no differences between sexes in the occurrence of this bacteria (Kaiser et al. 2010). According to our knowledge, there is no such data published concerning the infection rates for CPB. The closest to our scope was previously cited survey conducted by Hackett et al. (1996) in North America. Interestingly, Flavobacterium was reported to be a male-killing bacterium in Aphidophagous coccinellids (Majerus 2006). The infection with Flavobacterium was suggested to cause an intra-genomic conflict resulting from the cytoplasmic inheritance and to influence the reproductive strategy of infected insects favoring females survival, e.g., in Adalia bipunctata and Harmonia axyridis (Hurst et al. 1992, Majerus et al. 1998, Majerus 2003, Tinsley 2003). There is, however, no study addressing this phenomenon in the case of CPB so far.

The geographic location of the insect population can also be a factor influencing the endosymbiotic infection rate in insects. Flavobacterium was detected in all climatic zones (I–V) (Table 2, Fig. 1). The results of similar studies performed on other insect species show contradictory results depending on the insect studied. In Chinese populations of B. tabacii, the occurrence of endosymbiotic infections varied depending on the geographic location of the tested population of the insect (Pan et al. 2012). Also in Japanese populations of Curculio sikkimensis, local climate was the factor influencing the occurrence of endosymbiont infections (Toju and Fukatsu 2011). The infection frequencies of Sodalis, Wolbachia, and Rickettsia exhibited variations depending on temperature, precipitation, or snowfall (Toju and Fukatsu 2011). Again, according to our knowledge, there is no published data concerning described issues of CPB. But here because of the sample size, we cannot draw conclusion regarding an impact of geographic location on symbionts content in Polish CPB populations; however, we found Flavobacterium infection in CPB collected from all climatic zones in Poland.

Another factors influencing the endosymbiotic infection frequency is the biotype/haplotype of the tested insects and their host. All our tested insect individuals belonged to haplotype 1 (Jeszke et al. 2014).

L. decemlineata is a serious pest of potatoes and other solanaceous plants (Jacques and Fasulo 2010). Potato is a staple food consumed worldwide providing much of the world’s food supply. Poland is currently one of the biggest potato-growing countries in the world. Many varieties are developed and grown in Poland. For this reason, our study focused on the CPB populations collected from potato fields. However, research made on other insects revealed a significant correlation between the plant host and its insect pest in the endosymbiotic infection rate. For example, infection frequencies of the endosymbionts Serratia, Wolbachia, and Rickettsia, in insect populations of C. sikkimensis hosted on chestnuts Castanea crenata, were significantly higher than the same endosymbionts isolated from the insects hosted on Quercus serrata (Toju and Fukatsu 2011). The L. decemlineata is very well adapted to feeding on all Solanaceae plants (Jacques and Fasulo 2010). Potato is the preferred host for the CPB but CPB may also feed and survive on a number of other plants in the family Solanaceae, including belladonna, common nightshade, eggplant, ground cherry, henbane, horse-nettle, pepper (rarely), tobacco, thorn apple, tomato, and buffalo-bur. The CPB beetle has displayed an ability to adjust its host range to locally abundant Solanum species. These beetles also feed on non-solanaceous plants, but this is rare and these plants should not be considered as typical hosts (Capinera 2001).

The results of other researches, discussed above, showed that there was no one universal pattern for occurrence of endosymbionts in insects. They were present in all environments; however, the infection rates of the endosymbiotic bacteria tested for different insect hosts were reported higher when tested in the localizations with moderate climatic conditions. Despite the fact that Wolbachia is described as the most prevalent insect endosymbiont (Werren and Windsor 2000), we did not detect its presence in Polish CPB populations.

Summing up, in this study, we revealed a presence of Flavobacterium endosymbionts in Polish populations of L. decemlineata, mostly in male individuals and larva. To our knowledge, this is the first survey where CPB population was investigated for the presence of endosymbiotic species.