The characterization of Apis cerana and Apis mellifera mrjp2 gene in Indonesia and the phylogeny relationship with mrjp family genes

Karakterisasi gen mrjp2 Apis cerana dan Apis mellifera di Indonesia dan hubungan filogeni dengan gen mrjp famili

Abstract

The major royal jelly protein 2 (mrjp2) gene is one of the molecular markers that can discriminate between Apis cerana Fabricius and A. mellifera Linnaeus. Due to the lack of mrjp2 gene sequences registered in GenBank for Indonesian A. cerana and A. mellifera, DNA characterization and bioinformatics analysis were needed. This research aimed to characterize the exon-intron organization of mrjp2 genes for both Indonesian bee species and analyze the phylogenetic relationship with other mrjp family genes. DNA samples of Apis cerana and A. mellifera, collected from Bogor, were amplified using MF-MR primer at annealing temperatures of 47 ^oC and 50 ^oC, respectively. The length of A. cerana and A. mellifera DNA sequences were 579 and 597 bp, respectively. The DNA sequences of both species were comprised of partial exons 1, introns 1, exons 2, introns 2, and partial exon 3. The number of putative amino acids of A. cerana and A. mellifera mrjp2 genes were 111 and 123, respectively. We confirmed that the partial MRJP2 putative amino acids of both honey bees belong to the MRJP family and contained the peptide signal in the 14 first amino acid sites. Nucleotide variation of the mrjp2 gene in A. cerana was higher than A. mellifera. Phylogenetic tree construction showed that A. cerana and A. mellifera form a monophyletic clade with the A. mellifera mrjp7 gene and another mrjp family gene clustered as reported in a previous study.

INTRODUCTION

The major royal jelly protein (MRJP) is a group of proteins for royal jelly (Wang et al., 2020); these proteins are produced only by honey bees(Corzo et al., 2023). MRJP proteins are encoded by nine related genes: mrjp1-mrjp9 (Helbing et al., 2017). The mrjp family genes are grouped tandemly in a total of 60 kb base pairs and evolved from a single progenitor, i,e. the yellow protein gene family via gene duplication (Drapeau et al., 2006). This gene evolution conserves the exon/intron structure of the mrjp gene family (Drapeau et al., 2006). Although mrjp genes have similar exon/ intron structures, each gene produces a protein with unique characteristics and functions (Botezan et al., 2023). The molecular weights of MRJP1, MRJP2, MRJP3, and MRJP5 proteins are 53 kDa, 46.5 kDa, 66.8 kDa, and 80.9 kDa, respectively (A et al., 2022).

Mrjp genes encode proteins that are responsible for honeybee nutrition, physiological function, and the development of social behaviors such as caste determination (Buttstedt et al., 2013), as well as the division of labor for nursing and foraging among honeybee workers (Fang et al., 2023). The proteins of MRJP2 (Feng et al., 2021), MRJP8, and MRJP9 (Lee et al., 2022) show antimicrobial activities that are important for honey bee larval immune systems. Besides antimicrobial activity, the recombinants of Apis mellifera Linnaeus MRJP’s 1-7 provide antioxidant activity against H₂O₂(Park et al., 2020). Additionally, MRJP proteins have several health benefits, e.g, anti-tumor(Abu-Serie & Habashy, 2019), anti-aging (Jiang et al., 2018), anti-inflammatory (Hayashi et al., 2011), and antiapoptotic (Kim, 2021); summarized by (Mureşan et al., 2022).

The most common gene in the mrjp family is mrjp2 which encodes major royal jelly protein 2 (MRJP2) (Schmitzova et al., 1998). Mrjp2 gene sequences of Apis cerana Fabricius and A. mellifera show high polymorphism (Su et al., 2005). This gene was used to detect the entomological origin of A. cerana honey from China and A. mellifera honey from China, Brazil, Australia, and South Africa (Zhang et al., 2019) and also successfully distinguish the honey from those both bees in Java, Indonesia (Raffiudin et al., 2023). The first mrjp2 gene DNA sequences of A. cerana and A. mellifera originated from Indonesia and have been published in the GenBank (NCBI) database (Raffiudin et al., 2023). Another study found that there are three specific peptide markers of mrjp1, mrjp2, and mrjp3 genes for honey authentication, namely YNGVPSSLNVISK, TLQMIAGMK, and LTVAGESFTVK, respectively (Jiang et al., 2021).

Currently, the mrjp2 gene database at NCBI is limited to mRNA and Coding (CDS) sequences, with little information regarding exon/intron structure. Therefore, this study aims to characterize and analyze the exon/intron organization, nucleotide variation, variations in putative amino acids, and the phylogeny of A. cerana and A. mellifera bees in Indonesia based on their mrjp2 genes. Relationships with other mrjp family genes is also examined.

MATERIAL AND METHOD

Research locations

A total of 30 individuals from each one colony of A. cerana and A. mellifera honeybees were collected from beekeeping in Bogor, West Java by direct hand sampling method. All samples were paralyzed using 70% ethanol, and preserved in absolute ethanol at 4 °C until DNA extraction. This research was carried out at the Animal Molecular Laboratory, the Division of Animal Function and Behavior, Department of Biology, IPB University.

DNA extraction, amplification, and sequencing

Total DNA of each five individuals of A. cerana and A. mellifera honey bees were extracted from the thorax section based on the Phenol-Chloroform extraction method and ethanol precipitation (Sambrook et al., 1989) with modification (Raffiudin & Crozier, 2007). The DNA was diluted in 0.5 mM TE buffer and stored at 4 ℃. The target mrjp2 gene was amplified using MF-MR primer (Zhang et al., 2019). M-F primer ’-GCCATCCCTTGAAATTGTCACTCGT-3’) for forward and M-R primer 5’-GCCATCCCTTGAAATTGTCACTCGT-3) for reverse.

The MF-MR primer anneals at nucleotide positions 266 to 830 in the A. mellifera sequence (GeneID 406091), thus the target DNA comprised from exon 1 to exon 3 with an amplicon size target is ± 560 bp (Zhang et al., 2019). The annealing process was carried out at 47 ^ºC for A. cerana and 50 ^ºC for A. mellifera. DNA amplification was visualized using Diamond^TM Nucleic Acid Staining in Gel Doc and was sequenced by 1stBASE, Singapore service company.

The DNA sequences of the chromatogram samples were edited using BioEdit Sequence and Alignment (Hall, 1999) and Getenetyx-Win version 4.0.1 https://www.genetyx.co.jp/. The contig results were analyzed for homology using BLAST-N at the NCBI website (http://blast.ncbi. nlm.nih.gov/). The results of the sample DNA sequences were aligned with the DNA sequences of the mrjp2 gene of A. mellifera (406091) (Zhang et al., 2019) to determine the exon and intron organization using Clustal X software version 2.0 (Thompson et al., 1997). The intron positions were analyzed based on the GT/AG consensus patterns at the splice site (exons/introns) positions. Putative amino acid analysis was carried out using the Genetyx Win version 4.0.1 program. The exon positions were analyzed for nucleotide and putative amino acid variations using MEGA X software version 10.1.8 (Kumar et al., 2018). Protein signatures and families of MRJP2 putative amino acid sequences were explored using PROSITE (http://prosite.expasy.org/) and InterPro (https://www.ebi.ac.uk/interpro/), respectively. N-glycosylation in amino acids was predicted based on the study of (Lin et al., 2019). The schematic of the exon and intron mrjp family genes was constructed using http://wormweb.org/ exonintron.

Genetic distance and phylogenetic analysis

The phylogeny tree was constructed based on the nucleotide sequence of mrjp2 exons 2 and 3 according to the primer design. The resulting sequences, along with corresponding GenBank nucleotide sequences from A. cerana and A. mellifera, were aligned to determine homologyTable 1. The maximum likelihood (ML) method for generating phylogenetic trees was based on MEGA X software (version 10.1.8) with 1000 bootstrap replications (Kumar et al., 2018).

RESULTS

Homology and exon-intron organization of mrjp2 genes of Apis cerana and A. mellifera from Java

This study successfully characterized the 579 bp and 597 bp sequences of partial exon region 2 of mrjp2 genes from A. cerana and A. mellifera in Bogor, Java ( and, respectively). The homology analysis using online BLAST-N through NCBI (http://blast.ncbi.nlm.nih.gov/) showed that the A. cerana partial mrjp2 gene sequence has 99% query cover and 100% identity with A. cerana mrjp2 gene from Lebak Banten, East Java, Indonesia (LC596999.1)Table 2. Comparing A. mellifera from Bogor with A. mellifera from Bondowoso, East Java, sequence identity was 100% and query cover was 99% (LC600169.1)Table 3.

Both partial sequences of A. cerana and A. mellifera mrjp2 genes showed high AT percentages: 67% and 66%, respectively. Determination of exon and intron organization was carried out using the A. mellifera mrjp2 gene as a reference (Gen ID 406091). The resulting alignment showed that the DNA sequences of A. mellifera and A. cerana comprised partial exons 1, introns 1, exons 2, introns 2, and partial exon 3. The exon 2 region of A. cerana and A. mellifera yields 111. and 123 putative amino acids, respectivelyFigure 2, with introns 1 and 2 following the GT-AG consensus –. Based on protein family analysis using InterPro, the exon 2 and partial exon 3 putative amino acids of A. cerana and A. mellifera MRJP2 belong to the protein family of major royal jelly protein/protein yellow (IPR017996). The protein structure analysis using PROSITE https://prosite. expasy.org/ showed that the putative amino acids of A. cerana and A. mellifera MRJP2 from Bogor have signal peptides in the first 14 amino acid sites. The signal peptides of A. cerana partial MRJP2 are MTLWLFMVVCLGIA, while A. mellifera partial MRJP2 showed mutations in signal peptide at putative amino acid sites number 3 and 9. For amino acid number 3, lysine (K) in A. cerana is replaced by arginine (R) in A. mellifera . Similarly, amino acid number 9 of A. cerana is valine (V) which is replaced by alanine (A) in A. mellifera. Another protein signature reported by PROSITE is a lipid that is placed in the 15^th amino acid [Cysteine (C)] for both A. cerana and A. mellifera. Based on the previous study of N-glycosylation sites analysis (Lin et al., 2019), both A. cerana and A. mellifera showed the N-glycosylation site in the N₉₂ putative amino acid Figure 1–Figure 2.

Table 1.Apis mellifera and A. cerana sequences were used for phylogenetic analysis using mrjp family genes

No.	Gene name	Species	Accession number	Location	Molecular type	References
Ingroup
1.	mrjp2	A. mellifera	LC620983	Bogor, Indonesia	DNA	this study
2.	mrjp2	A. cerana	LC620984	Bogor, Indonesia	DNA	this study
3.	mrjp2	A. mellifera	NC_037648.1 (Gen ID 406091)	USA	Gene Whole Genome	(Wallberg et al., 2019)
4.	mrjp2	A. mellifera	NM_001011580	Germany	mRNA	(Dobritzsch et al., 2019)
5.	mrjp2	A. mellifera	AF000632	Germany	mRNA	(Schmitzova et al., 1998)
6.	mrjp2	A. mellifera	GQ160519	South Korea	mRNA	Yoon & Nguyen et al. 2009 (unpublished)
7.	mrjp2	A. mellifera carnica	KX951418	Germany	CDS	(Helbing et al., 2017)
8.	mrjp2	A. mellifera	XM_026443530	USA	mRNA isoform X1	(Wallberg et al., 2019)
9.	mrjp2	A. cerana	NW_016019131 (Gen ID 107997173)	South Korea	mRNA	(Park et al., 2019)
10.	mrjp2	A. cerana	AF525777	Thailand	mRNA	Sittipraneed et al. 2004 (unpublished)
11.	mrjp2	A. cerana	AY392758	China	mRNA	(Su et al., 2005)
12.	mrjp2	A. cerana	AY515689	Thailand	CDS	(Imjongjirak et al., 2005)
13.	mrjp2	A. cerana	MH551225	South Korea	mRNA	(Park et al., 2019)
14.	mrjp7	A. mellifera	NM_001014429.1	*	DNA	(Elsik et al., 2014)
15.	mrjp5	A. mellifera	GU339164.1	*	mRNA	Yoon & Nguyen 2009 (unpublished)
16.	mrjp5	A. mellifera	NM_001011599.1	Germany	mRNA	(Dobritzsch et al., 2019)
17.	mrjp5	A. cerana	AY392757.1	China	mRNA	(Su et al., 2005)
18.	mrjp5	A. cerana	NM_001328480.1	*	mRNA	Sittipraneed et al. 2004 (Unpublished)
19.	mrjp3	A. mellifera	NM_001011601.1	Germany	mRNA	(Dobritzsch et al., 2019)
20.	mrjp6	A. mellifera	NM_001011622.1	*	DNA	(Elsik et al., 2014)
21.	mrjp1	A. cerana	AY279539.1	China	mRNA	(Su et al., 2005)
22.	mrjp1	A. mellifera	GQ160518.1	*	mRNA	Yoon dan Nguyen 2009 (unpublished)
23.	mrjp1	A. mellifera	NM_001011579.1	Germany	mRNA	(Dobritzsch et al., 2019)
24.	mrjp4	A. cerana	MF402924.1	South Korea	mRNA	(Kim et al., 2019)
25.	mrjp4	A. mellifera	GU325612.1	South Korea	mRNA	Yoon & Nguyen 2010 (unpublished)
26.	mrjp4	A. mellifera	NM_001011610.1	*	DNA	(Elsik et al., 2014)
27.	mrjp8	A. mellifera carnica	EU703874.1	*	mRNA	(Peiren et al., 2008)
28.	mrjp8	A. mellifera	NM_001011564.2	Brazil	mRNA	(Parpinelli et al., 2014)
29.	mrjp9	A. mellifera	EU703875.1	*	mRNA	(Peiren et al., 2008)
30.	mrjp9	A. mellifera	NM_001024697.1	*	mRNA	(Elsik et al., 2014)
31.	mrjp9	A. mellifera	DQ000307.1	*	mRNA	(Albert & Klaudiny, 2007)
31.	mrjp9	A. mellifera	DQ000307.1	*	mRNA	(Albert & Klaudiny, 2007)
Outgroup
32.	mrjp2	Bombus impatiens	NT_177730	USA	DNA Whole Genome	Robertson 2015 (unpublished)

Nucleotide and putative amino acid variation of A. cerana and A. mellifera partial mrjp2 genes

Nucleotide variation analysis was carried out based on the 223 bp of exon 2 and 111 bp of exon 3. The results showed that in A. cerana there were 14 and 16 nucleotide variations in exons 2 and 3, respectivelyTable 4. However, the A. mellifera mrjp2 gene is more conserved, with only three nucleotide variations in exon 3. Nucleotide variations between A. cerana and A. mellifera occurred in 43 sites with more variation exon 2 than exon 3. Higher nucleotide variation in A. cerana resulted in higher variation in putative amino acids. A. cerana has 15 while A. mellifera has but one. The one mutation in A. mellifera occurs at site 96 in exon 3 of the mrjp2 gene changing the amino acid glycine (G) to aspartic acid (D)Table 5.

Genetic distance and phylogeny of mrjp genes

The genetic distance of the mrjp genes of A. cerana and A. mellifera was analyzed based on exon regions 2 and 3 using the Tamura 3-parameter analysis model (Nei & Kumar, 2000). The genetic distance within A. cerana ranges from 0.0030 to 0.1132, while lower ranges of 0.000 to 0.0091 were observed in A. mellifera (Supplementary 1). Thus, the genetic distance between both honey bee mrjp2 genes ranged from 0.0799 to 0.1015. The highest genetic distance within all A. cerana and A. mellifera mrjp genes (mrjp1-9) was 0.5479, which occurred from A. mellifera mrjp1 and mrjp9 gene (Supplementary 1).

Although the schematic structure of the mrjp family genes show that mrjp1 to mrjp9 have varied lengths and exon-intron structures, almost all coding regions (black boxes) of these gene have the similar patterns. Construction of an ML phylogenetic tree for A. cerana and A. mellifera mrjp family genes (mrjp1-mrjp9) revealed the presumptive evolution of mrjp genes. Sequences were clustered based on the gene with bootstrap values greater than 75%. All A. cerana and A. mellifera mrjp2 genes are clustered in a single clade along with the mrjp7 gene (bootstrap results of 77%,). As shown in, despite lower bootstrap measures, the mrjp2+7 gene clade was clustered with mrjp1 (40% bootstraps). The mrjp4 gene is the sister clade of mrjp2+7 and mrjp1 (34% bootstraps). The mrjp6+mrjp3 clade was related to mrjp5 (18% bootstraps). Finally, the mrjp8+mrjp9 gene clade was placed at the base of the tree (41% bootstraps).

Table 2.BLAST-N results of Apis cerana partial mrjp2 gene sequences from Bogor

Description	Query cover	E value	Identity value	Accession number
A. cerana mrjp2 gene Lebak Banten, partial cds	99%	0	100.00%	LC596999.1
A. cerana mrjp2 gene Bantul, partial cds	99%	0	99.83%	LC600197.1
A. mellifera carnica mrjp2 gene, complete cds	99%	0	90.89%	KX951418.1

Table 3.BLAST-N results of Apis mellifera partial mrjp2 gene sequences from Bogor

Description	Query cover	E value	Identity value	Accession number
A. mellifera mrjp2 gene Bondowoso, partial cds	99%	0	100.00%	LC600169.1
A. cerana carnica, complete cds	100%	0	98.99%	KX951418.1
A. cerana mrjp2 (mrjp1) gene, complete cds	96%	0	90.35%	AY515689.1

Figure 1.Structure of nucleotide sequences (up) and deduced amino acid (down) of partial mrjp2 gene in Apis cerana Bogor (LC620984). The numbering on the right indicates the position of the last nucleotide (up) or amino acid (down) in each line. Deduced amino acids with underline and box are signal and lipid-based on PROSITE, respectively. Deduced N-glycosylation in amino acids based on Lin et al. (2019) is indicated by an asterisk.

Figure 2. Structure of nucleotide sequences (up) and deduced amino acid (down) of partial mrjp2 gene in Apis mellifera Bogor (LC620983). The numbering on the right indicates the position of the last nucleotide (up) or amino acid (down) in each line. Deduced amino acids with underline and box are signal and lipid-based on PROSITE, respectively. Deduced N-glycosylation in amino acids based on Lin et al. (2019) is indicated by an asterisk.

Table 4.Apis mellifera and A. cerana’s nucleotide variations of mrjp2 gene in exon 2 and 3 region

No.	Sequences	Nucleotide site-																									Nucleotide site-
		Exon 2																									Exon 3
														1	1	1	1	1	1	1	1	1	2	2	2	2	2	2	2	2	2	2	3	3	3	3	3	3	3	3	3	3	3	3
			1	2	3	3	6	7	8	8	8	9	9	0	1	2	3	4	4	5	5	9	0	2	4	6	6	6	6	8	8	9	0	0	0	1	1	1	1	2	3	3	4	5
		8	8	6	3	6	0	4	0	7	9	5	6	9	2	2	6	0	2	1	4	9	2	6	0	0	3	7	8	0	7	6	5	6	7	2	3	6	9	8	2	5	0	3
1.	A. mellifera mrjp2 NM001011580	G	C	C	C	C	C	G	G	C	C	A	G	A	A	A	G	T	T	C	C	T	C	C	G	A	C	G	A	T	A	G	G	G	T	C	T	T	G	A	T	A	A	T
2.	A. mellifera Bogor mrjp2 exon 2 3 LC620983*	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
3.	A. mellifera mrjp2 GQ160519.1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
4.	A. mellifera mrjp2 AF000632.1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
5.	A. mellifera mrjp2 KX951418.1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	G	.	.	.	A	.	.	.	.	.	.	C	.	.	.
6.	A. mellifera mrjp2 XM026443530.1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	G	.	.	.	A	.	.	.	.	.	.	C	.	.	.
7.	A. mellifera mrjp2 406091	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	G	.	.	.	A	.	.	.	.	.	.	C	.	.	.
8.	A. cerana mrjp2 AY392758	A	T	.	T	.	T	A	C	.	G	.	A	T	G	.	A	A	C	T	.	C	T	T	A	.	.	A	G	G	.	A	A	A	C	T	C	.	.	C	C	C	.	.
9.	A. cerana mrjp2 AY515689	A	T	.	T	.	.	A	C	.	G	.	A	T	G	.	A	A	C	T	.	C	T	T	A	.	.	A	G	G	.	A	A	A	C	T	C	.	.	C	C	C	.	.
10.	A. cerana mrjp2 AF525777.2	.	.	.	T	.	.	A	C	.	G	.	A	T	G	.	A	A	C	T	.	C	T	T	A	.	.	A	G	G	.	A	A	A	C	T	C	.	.	C	C	C	.	.
11.	A. cerana mrjp2 MH551225.1	.	.	.	T	G	T	A	C	.	G	.	A	T	.	.	A	A	C	T	.	C	T	T	A	.	.	A	G	G	.	A	A	A	C	T	C	.	.	C	C	C	.	.
12.	A. cerana mrjp2 GenID 107997173	A	T	T	T	.	.	.	A	T	G	C	.	T	G	C	A	A	C	T	T	C	T	T	.	G	T	.	.	G	T	.	.	A	.	.	.	C	C	.	C	C	G	.
13.	A. cerana Bogor mrjp2 exon 2 3 LC620984*	A	T	T	T	.	.	.	A	.	G	C	.	T	G	C	A	A	C	T	.	C	T	T	.	G	T	.	.	G	T	.	.	A	.	.	.	C	C	.	C	C	.	C

DISCUSSION

Both mrjp2 gene sequences of A. cerana and A. mellifera bees have higher AT nucleotide content than GT nucleotide content, consistent with (Raffiudin et al., 2022). The whole genome A. mellifera also reported that honey bees contain high amounts of adenine (A) and thymine (T) compared to other insect genome sequences (Honeybee Genome Sequencing Consortium 2006). The results of the BLAST-N DNA sequences of A. cerana and A. mellifera showed that both were homologous to the samples of A. cerana (LC596999.1) and A. mellifera (LC600169.1) from Java, Indonesia with 100% homology values and an E-value of 0. A nucleotide sequence can be said to be homologous if the results of homology analysis using BLAST-N show an E-value close to zero (Pearson, 2013)or an identity value greater than 70% for nucleotide sequence data and more than 25% for the analyzed amino acid sequence data (Claviere & Notredame, 2007). The high homology of A. cerana is consistent with the previous results that found there is no variation of the mrjp2 gene in A. cerana from several provinces in Indonesia (Raffiudin et al., 2022)

Alignment results showed that A. cerana and A. mellifera sequences comprised the exon 1 to partial exon 3, this result confirmed the targeted mrjp2 region of MF-MR primer (Zhang et al., 2019). The intron region of A. cerana and A. mellifera mrjp2 gene sequences have fulfilled the GT-AG consensus (Kitamura-Abe et al., 2004), that flank the exon region (Breathnach et al., 1978). The resulting of 112 and 123 putative amino acids in exon 2 and 3, respectively of both honey bee were confirmed by using InterPro as the MRJP family member. The MRJP protein contains around 400–578 putative amino acids (Buttstedt et al., 2013) with the mrjp2 gene having six exons and five introns (Drapeau et al., 2006). This study found that the first 14 putative amino acids were detected as the N-terminal signal peptide both in A. cerana and A. mellifera. This signal is supported by the study that revealed the MRJP family consists of 16–20 amino acids N-terminal signal peptide (Schmitzova et al., 1998); (Buttstedt et al., 2014). A recent study found that there were three N-glycosylation sites in MRJP2 isolated from fresh royal jelly of A. mellifera, which are N145, N178, and N₉₂ (Lin et al., 2019). Based on (Lin et al., 2019), our result confirmed that the MRJP2 putative amino acid of A. cerana and A. mellifera showed the expected N-glycosylation site in the asparagine (N) amino acid number 92. The N-glycosylation has been conserved and is important since these asparagine residues are the attachment sites for complex sugars or glycosylation (Park & Zhang, 2011).

Variations between A. cerana and A. mellifera in their nucleotide sequences and putative amino acids may reflect differences in the quantity and composition of the MRJP proteins that make up royal jelly (Yu et al., 2009). Moreover, the results show that A. cerana has more within-species nucleotide and amino acid variation than A. mellifera. This high genetic variation in A. cerana nucleotide sequences might be due to a wider geographical distribution and the presence of different subspecies. Variation within subspecies of A. cerana in Indonesia is low (Raffiudin et al., 2022).

The genetic distance results show that A. cerana bee samples have a genetic distance that was closer to the DNA sequences of the A. cerana mrjp2 gene than to the DNA sequences of the A. mellifera mrjp2 gene (Supplementary 1). This result follows (Su et al., 2005) that the mrjp2 gene of A. cerana and A. mellifera has a large polymorphism.

Although the mrjp2 gene of A. cerana and A. mellifera have a high polymorphism, however, they formed the monophyletic clade. Uniquely, the phylogenetic topology of A. cerana based on exons 2 and 3 of the mrjp2 gene showed that A. cerana from Bogor clustered with A. cerana (Gen ID 107997173) with a bootstrap value of 77%. This clade was separate from the other mrjp2 genes of A. cerana and A. melliferaFigure 4. Furthermore, the topology of the mrjp family gen tree showed that the A. cerana and A. mellifera mrjp2 clustered with the A. mellifera mrjp7 gene with a low bootstrap value of 41%Figure 4. The entire mrjp2 and mrjp7 family genes in the phylogenetic tree can still be classified as one cluster because the resulting bootstrap value is relatively lowFigure 4. Clusters in a phylogenetic tree are said to be real and reliable if they have a bootstrap proportion value of more than 70% (Hillis & Bull, 1993). The clustering of mrjp2 and mrjp7 genes are congruent with the phylogenetic tree of Apis spp. using the mrjp family that showed mrjp2 is grouped in the same cluster as mrjp7 (Drapeau et al., 2006); (Buttstedt et al., 2014).

The mrjp family gene tree also revealed that mrjp3+mrjp6 and mrjp5 genes were closely related. The mrjp3+mrjp6 group is similar to (Buttstedt et al., 2014) study, while the position of mrjp5 is inconsistent (Drapeau et al., 2006). On the other hand, mrjp8 and mrjp9 were clustered forming a group that is separated from other mrjp family membersFigure 4. This is consistent with previous studies (Drapeau et al., 2006); (Buttstedt et al., 2014). Perhaps the functions of mrjp8 and mrjp 9 differ from mrjp1-7. Unlike other mrjp family genes, the expression of mrjp8 and mrjp9 were low both in the brain and hypopharyngeal gland, while expressed in another organ, leading to the hypothesis that the function of those genes differs from other mrjp genes(Dobritzsch et al., 2019). The gene sequence of the mrjp families A. cerana and A. mellifera can group in the same cluster because the mrjp family genes evolved from one gene of the same family. The mrjp gene family of Apis spp. evolved from a common background, thus they have a close relationship (Drapeau et al., 2006). The mrjp gene family forms a large cluster with the mrjpl/yellow gene family in the Hymenopteran species, which includes Apis honey bees, non-Apis bees, and ants (Buttstedt et al., 2014). Knowledge of the mrjp gene family’s structure and evolution is important for our understanding of their roles in honey bee development and nutrition.

Table 5.Apis cerana and A. mellifera putative amino acid variations of exon 2 and 3 regions of mrjp2 gene

No.	Species	Putative amino acid site-
		Exon 2											Exon 3
																			1	1
				2	2	2	2	2	3	3	4	7	8	8	9	9	9	9	0	0
		3	9	0	2	4	5	6	0	5	1	4	1	3	0	3	6	8	0	6
1.	A. mellifera mrjp2 NM001011580	R	A	V	E	S	P	R	K	I	F	R	I	R	T	V	G	T	K	K
2.	A. mellifera Bogor mrjp2 exon 2 3 LC620983*	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
3.	A. mellifera mrjp2 GQ160519.1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
4.	A. mellifera mrjp2 AF000632.1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
5.	A. mellifera mrjp2 KX951418.1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	D	.	.	.
6.	A. mellifera mrjp2 XM026443530.1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	D	.	.	.
7.	A. mellifera mrjp2 406091	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	D	.	.	.
8.	A. cerana mrjp2 AY392758	K	.	I	Q	.	A	K	N	.	I	H	.	K	.	M	N	I	.	Q
9.	A. cerana mrjp2 AY515689	K	.	I	Q	.	A	K	N	.	I	H	.	K	.	M	N	I	.	Q
10.	A. cerana mrjp2 AF525777.2	.	.	I	Q	.	A	K	N	.	I	H	.	K	.	M	N	I	.	Q
11.	A. cerana mrjp2 MH551225.1	.	.	I	Q	.	A	K	N	.	I	H	.	K	.	M	N	I	.	Q
12.	A. cerana Bogor mrjp2 exon 2 3 LC620984*	K	V	.	K	.	A	.	N	L	I	.	V	.	S	.	D	.	N	Q
13.	A. cerana mrjp2 GenID 107997173	K	V	.	K	F	A	.	N	L	I	.	V	.	S	.	D	.	N	Q

Figure 3.Schematic position of Apis mellifera (LC620983) and A. cerana (LC620984) partial mrjp2 gene in Bogor compared to A. mellifera mrjp gene family. The box indicates the exon region and the line indicates the intron. Scale 100 bp.

Figure 4.The nucleotide sequence-based phylogenetic tree of mrjp family genes in Apis cerana and A. mellifera using the asterisks code (*) indicates current samples.

CONCLUSION

This study successfully isolated 579 bp and 597 bp of mrjp2 genes from A. cerana and A. mellifera, respectively. Those sequences are homologs with the mrjp2 gene of A. cerana and A. mellifera from Indonesia. They are comprised of partial exons 1 up to partial exon 3, and produce 111 and 123 putative amino acids of A. cerana and A. mellifera, respectively. Nucleotide and putative amino acid variation in A. cerana is higher than in A. mellifera. Phylogenetic tree construction showed that the A. cerana and A. mellifera mrjp2 genes have a close phylogenic relationship with the A. mellifera mrjp7 gene. The structure and phylogenetic relationships confirm that this family of genes has conserved exon/intron structure through gene duplication.

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