Genetic diversity among queen bee, worker bees and larvae in terms of retrotransposon movements

Honey bee (Apis mellifera L.) is a model organism, contributing significant effect on global ecology by pollination and examining due to its social behaviour. In this study, barley-specific Sukkula and Nikita retrotransposons were analysed using IRAP (Inter-Retrotransposon Amplification Polymorphism) marker technique, and the relationships between retrotransposon movements and development were also investigated in three different colonies of the Caucasian honey bee (A. mellifera caucasica). Furthermore, transposon sequences belonging to A. mellifera, Bombus terrestris, Triticum turgidum L. and Hordeum vulgare L. were also examined to figure out evolutionary relationships. For this purpose, a queen bee, five worker bees, and five larvae from each colony were studied. Both retrotransposons were found in all samples in three colonies with different polymorphism ratios (0–100% for Nikita and 0–67% for Sukkula). We also determined polymorphisms in queen–worker (0–83% for Nikita, 0–63% for Sukkula), queen–larvae (0–83% for Nikita, 0–43% for Sukkula) and worker–larvae comparisons (0–100% for Nikita, 0–63% for Sukkula) in colonies. Moreover, close relationships among transposons found in plant and insect genomes as a result of in silico evaluations to verify experimental results. This work could be one of the first studies to analyse plant-specific retrotransposons’ movements in honey bee genome. Results are expected to understand evolutionary relationships in terms of horizontal transfer of transposons among kingdoms.


Introduction
The vast majority of all known animal species are members of the Arthropoda phylum, and the class Insecta is the most significant class of Arthropods. Almost 80% of animal species defined in the world are found in the class Insecta (Stork et al. 2015;Zhang 2013). Honey bee (A. mellifera L.) is an economically and ecologically critical social insect for human beings. In the beehive, female bees (queen and worker) originate from fertilised eggs and males (drone) from unfertilised eggs. If larvae are specially fed with royal jelly, it turns into queen bees and produces thousands of eggs in a day while if not feed with royal jelly, they become sterile workers. Therefore, the bee is a very suitable model organism to study the relationship between nutrition (environment) and genetic structure (The Honey Bee Genome Sequencing Consortium 2006).
Transposons, mobile genetic elements, are divided into two groups including Class I (RNA transposons or retrotransposons) and Class II (DNA transposons). They move via copy-paste and cut-paste mechanism, respectively. They constitute different portions of animal genomes (3-60%). This ratio is very high in plant genomes (up to 90%) (Canapa et al. 2015). It is reported that the genome size of the honey bee is 236 Mb, and approximately 3% of the genome consist of transposons. Although there are many retrotransposons in the honey bee genome, there are little pieces of evidence for active transposable elements (The Honey Bee Genome Sequencing Consortium 2006).
In this study, we aimed to investigate the presence and movements of barley-specific Nikita and Sukkula retrotransposons by using IRAP marker technique and evaluation the polymorphisms in different colonies at different developmental stages in honey bee genome for the first time. Moreover, we obtained transposon sequences belonging to different genomes from NCBI to figure out the evolutionary relationships.

Honey bee materials
In this study, three colonies of Apis mellifera caucasica were selected from the beekeeping unit of Department of Animal Sciences of Faculty of Agriculture in Ondokuz Mayıs University (41°21 0 37.2 00 N 36°11 0 12.8 00 E). Genomic DNA of a total of 33 honey bee samples consisting of 11 samples from each colony (1 queen, 5 workers, and 5 larvae) were isolated according to Evans et al. (2013). The concentration and quality of genomic DNA were determined using a spectrophotometer and 1% (w/v) agarose gel.

IRAP analyses
Barley-specific Nikita and Sukkula retrotransposons' movements were analysed by IRAP molecular marker technique.
Primer sequences were 5 0 ACCCCTCTAGGCGACATCC3 0 for Nikita and 3 0 GGAACGTCGGCATCGGGCTG5 0 for Sukkula (Leigh et al. 2003). IRAP-PCR was carried out in 25 lL of the reaction mixture containing 6.5 lL of nuclease-free dH 2 O, 12.5 lL of MasterMix (Dream-Taq Green PCR Master Mix, Thermo Scientific TM ), 2 lL of 10 lM/lL primer (0.8 lM/lL), 4 lL of 20 ng/ lL (3.2 ng/lL) template genomic DNA. Final concentrations were indicated in parentheses. The amplification conditions were as follows: one initial denaturation step (95°C for 3 min) followed by 35 cycles of denaturation (95°C for 30 s), annealing (54°C for 30 s) and extension (72°C for 1 min). The reaction was completed by a final extension step (72°C for 5 min). PCR products were resolved on 1% agarose gel (w/v) in 1X TBE (Tris-Boric acid-EDTA) at 120 V for 120 min. Then, agarose gel photographed on a UV transilluminator and band profiles belonging to samples were evaluated.

Calculations of polymorphism
Band profiles of queen bees, worker bees, and larvae were examined one by one for each sample, and monomorphic and polymorphic bands were determined. Polymorphism rates among samples were calculated using the Jaccard similarity index. Jaccard's similarity index could be calculated using the formula: N AB /(N AB ? N A ? N B ); where N AB is the number of bands shared by two samples, N A represents amplified fragments in sample A, and N B represents amplified fragments in sample B (Jaccard 1908).

Evolutionary relationships among transposons
Transposon sequences of related species Apis mellifera and Bombus terrestris in addition to Nikita and Sukkula sequences belonging to Triticum turgidum L. and Hordeum vulgare L. were examined to evaluate evolutionary relationships among these species and verify our IRAP results. For this purpose, sequences were retrieved from NCBI (www.ncbi.nlm.nih.gov) and a phylogenetic tree was constructed by using these sequences via MEGA X (Kumar et al. 2018) with following parameters: neighbour-joining (NJ) method (Saitou and Nei 1987) with the p-distance model (Nei and Kumar 2000), and even bootstrap test (1000 replicates) (Felsenstein 1985).

Nikita showed higher polymorphism than Sukkula
Barley-specific Nikita retrotransposon was identified in queen bees, worker bees, and larvae. As a result of IRAP-PCR, samples displayed a band profile in the range of 200-1500 bp (Fig. 1).
Moreover, polymorphism rates were also detected within colonies. Polymorphism rates of queen-workers and queen-larvae were the same in the first colony (0-71%). Polymorphism ratio was also determined as 14-71% for workers-larvae. In the second colony, there was 0-50% polymorphisms for queen-workers, 0-57% for queen-larvae. However, this rate increased by 67% for workers-larvae. The most polymorphic patterns were determined in the third colony. Polymorphism rates for queen-worker and queen-larvae were found to be the same but higher (0-83%) compared to other colonies. Besides, 0-100% of polymorphism rates were determined as a result of comparison between workers and larvae.
Similar to Nikita, barley-specific Sukkula retrotransposon was also identified in the queen, workers and larvae, ranging from 250 to 1500 (Fig. 2). When compared to Nikita, Sukkula indicated lower polymorphism percentages among samples.
In general, polymorphism rates among individuals of all colonies for Sukkula retrotransposon were determined in the range of 0-67%. However, polymorphism rates among samples in the first colony were 0-43% while 0-63% for the second and third colonies. When compared to queens, workers and larvae in three colonies, 14-57%, 20-50% and 0-43% ratios were observed, respectively ( Table 2).
The highest polymorphism rates were identified for queen-larvae (43%) in the first colony. Also, there was 20% polymorphism for queen-workers and 29% for worker-larvae. In the second colony, the lowest polymorphism rate was observed for queen-larvae comparison (14%). On the other hand, higher polymorphism was detected for queen-workers (33-57%) and workers-larvae (0-63%). These rates were 0-38% for queen-larvae, 43-63% for queen-workers and 20-63% for workers-larvae in the third colony.
In silico analyses showed similarities among retrotransposons in insect and plant genomes A phylogenetic tree was constructed by using 53 different sequences (Fig. 3). All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 105 positions in the final dataset. We observed that all sequences were clustered into two groups. Most of A. mellifera     sequences were found in the first group. Moreover, A. mellifera and B. terrestris sequences were found in one clade while Sukkula sequences formed a second clade. In the second group, similar to the first one, A. mellifera, B. terrestris and Sukkula sequences were observed. In addition, Nikita sequences were also determined in this group.

Discussion
Transposable elements can be inherited via vertical transfer which is genetic transfer from ancestral to descendant species (Markova and Mason-Gamer 2015;Wallau et al. 2016). On the other hand, many evidence also showed that these sequences move horizontally (Melo and Wallau 2020;Zhang et al. 2020;Wanner and Faulk, 2021). Phylogenetic relatedness among species is an important factor for horizontal transfer of transposons (HTT). There are many studies in which a retrotransposon specific to a particular plant is detected in a different plant. In one of these studies, Nikita and Sukkula retrotransposons were studied in the aniseed. Polymorphism was not found in both retrotransposons but their presence in the aniseed genome was determined (Marakli 2018). In another study, these two retrotransposons were identified in two varieties of black pine (Pinus nigra var. pyramidata and Seneriana). Sukkula retrotransposon showed 0-76% polymorphism in pyramidata variety while no polymorphism was detected in Seneriana variety. Similarly, Nikita polymorphism (0-56%) was detected only in pyramidata variety (Marakli et al. 2019). Bagy2 (0-73%) and Nikita (37-100%) polymorphism ratios were also investigated in GM and non-GM soybean grown under salt stress (Sahin et al. 2020). Moreover, Safiyar et al. (2021) studies Aegilops tauschii accessions and its relatives, tetetraploid and hexaploid wheat by using barley-specific retrotransposons via both IRAP and REMAP molecular markers.
In addition to closely related species, HTT could also observe in distantly related taxa (Gao et al. 2018;Metzger et al. 2018;Zhang et al. 2020). Concordant with this opinion, we identified barley-specific retrotransposons in chicken genome for the first time in our previous study (Mercan et al. 2022). In this presented study, barley-specific Nikita and Sukkula movements were also analysed by using IRAP marker technique in bees with different growth capabilities due to feeding in different periods. Higher polymorphism ratios were determined in bee genome (0-100% for Nikita and 0-67% for Sukkula) among queen, workers and larvae. Moreover, similar sequences were identified among transposons in Apis mellifera, Bombus terrestris, Triticum turgidum and Hordeum vulgare genomes. These results could be supported by Meyerowitz (2002), reporting a common ancestor for plants and animals.
In honey bee genome, there are very few transposons including 15 partial sequences of a Copia family sequence with highly distorted copies, 6 partial sequences matching the coding BEL12 element of Anopheles gambiae, and 3 highly degraded copies of a DIRS retrotransposon (Eiglmeier et al. 2005;Goodwin et al. 2004). Moreover, 11 LTR and 7 non-LTR retrotransposon residues in Drosophila were also found in honey bee genome (Kaminker et al. 2002). Beye et al. (2006) suggested that all transposons determined in bee genome belong to mariner family with * 360 copies in the genome. Other study was carried out by Gillespie et al. (2006). They suggested that ribosomal DNA units of honey bees contain active non-LTR retrotransposons of the R2 family. They also reported that although active R2 non-LTR retrotransposons were identified, R1 line was not found in honey bee genome. Elsik et al. (2014) also determined the presence of copia, R2 and

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I retrotransposons together with mariner and piggyBac transposons in the genome of the honey bee. Similarly, Wang et al. (2017) reported that queen bees and drones contain mariner, piggyBac, R2, copia, Bel-Pao and I transposons but worker bees only contain mariner, piggyBac and R2 transposons. There are many studies related to the effect of retrotransposon movements on species identification (Leśniowska-Nowak et al. 2021), organ differentiation (Ramos et al. 2011;Zhu et al. 2012) and abiotic/biotic stress (Ghonaim et al. 2021) conditions by using retrotransposon-based molecular markers. Most of these studies were performed in plant genomes because transposons constitute most of plant genomes.

Conclusions
The honey bee is a crucial component of the ecological balance and lives a mutually dependent life on vegetation. The physiological development of individuals in the hive depends on their nutrition including royal, worker, and drone jellies, changing gene expression profile of larvae. Therefore, there has been a critical importance in terms of the relationships between plants' genome and honey bee genome to identify molecular mechanisms under the feeding and development. This study provided evidence that the honey bee genome is more dynamic than predicted. To our best knowledge, there is no investigation related to plant-specific retrotransposons in the honey bee. The findings could be preliminary results to understand the difference between Nikita and Sukkula retrotransposons' movements related to retrotransposon effects on an important species for humanity's future. Thus, further studies are needed to determine the relationship between gene expression profiles and retrotransposons depending on different environmental conditions. Moreover, deep sequencing of honey bee genome will provide a better understanding of the relationships among different organisms in different species.