Transcriptomic comparison of the royal jelly proteins coded in the hypopharyngeal glands of Apis mellifera and Geotrigona acapulconis

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Transcriptomic comparison of the royal jelly proteins coded in the hypopharyngeal glands of Apis mellifera and Geotrigona acapulconis

https://doi.org/10.21203/rs.3.rs-2948844/v1

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Apis mellifera and Geotrigona acapulconis are two members of Apidae family with large morphological differences; that is G. acapulconis is stingless, smaller in size, lack of yellow pigmentation, different nest building and hive structure, among other differences. We also suggest that there are biochemical differences between A. mellifera and G. acapulconis related to the composition of their Royal Jelly proteins (RJPs) and Yellow family proteins (YFPs). For instances, it has been reported that RJPs are involved in larval development of A. mellifera, and the YFPs play an important role in pigmentation in these arthropods. In this work, the protein composition of RJPs and YFPs were investigated indirectly by performing transcriptome analysis of the hypopharyngeal glands (HG), which are the RJP and YFPs producers. The results showed the presence of transcripts coding for RJPs in both bees. These proteins belonging to the Major Royal Jelly proteins (MRJPs) only recorded in A. mellifera and YFPs, which were identified here in both bees, but in different proportions. Additionally, MRJPs were only found in A. mellifera. Yet, coding sequences of these type proteins were found as Royal Jelly protein-like (RJPL) in G. acalpuconis, which are evolved from the MRJP. Furthermore, the differences found between yellow proteins (YPs) could be directly associated to the color of such bees, and the lack of MRJPs in G. acapulconis, that are important for larval nutrition and development, could be a consequence of a poor development of the larvae. On the other hand, some of the differences could be the result of their different behavior. Some MRJPs are required for defense of the larvae against pathogens, which are related to the differences in their biological cycle. Unfortunately, other functions of RJPs are not yet fully characterized; so, it is imperative to continue the research of the functions of RJPs in bees for a better understanding of their biological role.

Geotrigona acapulconis

Apis mellifera

transcriptome

RNA-Seq

hypopharyngeal glands

The Apidae family include the honeybee Apis mellifera, in which ten yellow-MRJPs (yellow-MRJP1-9 plus a pseudogene) have been found (Ferguson et al., 2011) and the stingless bee Geotrigona acapulconis (Strand, 1919), Apis mellifera is the most worldwide and studied bee, and G. acapulconis (endemic of America), which is a bee that was described firstly in Mexico (Ala, 1999), but according to the Global Biodiversity Information Facility (GBIF, https://www.gbif.org) some related species have been also observed in Costa Rica and have also been reported in other LATAM countries such as Brazil (Arnold et al., 2018) and Colombia (Gonzalez et al., 2012). In Mexico, the G. acapulconis species is mainly found from the Balsas River Basin to the Isthmus of Tehuantepec (Ala, 1999); even though there is little information about biochemical, physical, or behavioral characteristics of this bee (Can-Alonzo et al., 2005; Insuasty-Santacruz et al., 2017; Quezada-Euán et al., 2018). Despite both bees are from the same family, the most remarkable differences between them lies in their morphology, followed by the structure of the hive, the biological cycle, and the number of queens (Arnold et al., 2018). The most notable morphological differences between them are the fact that G. acapulconis is a stingless bee, but not less important they had noticeable differences in size and pigmentation. Regarding to the differences in the hive, G. acapulconis has a nest in tree cavities, in the ground, in termite mounds, and their ridges are built in horizontal layers (Inoue et al., 1984). On the contrary, A. mellifera build their nests in the air, between branches or walls, which can be exposed or semi-exposed, and their combs are built on pillars (Kilani, 2005). The last known difference is that stingless bees have a fertilized queen, as do honeybees, but unlike honeybees, they have several virgin queens (Arnold et al., 2018.). The number of queens varies between species (Hartfelder et al., 2006). In relation with the hypopharyngeal glands (HG), they have been described in queens and worker bees (Li et al., 2008). These glands are responsible to produce the royal jelly that worker bees will give to the larval and the queen as food (Page et al., 1998). The HG consist of a pair of glands on the head which are long, sinuous tubes to which solid circular lobes are attached (Ilyasov et al., 2020). The secretion is milky and pasty, whitish in color, with a pronounced acidity (Ilyasov et al., 2020). This substance is administered to all the larvae during the first three days of growth, but after the fourth day on, pollen and honey are added to continue their growth towards bee workers or drones (Li et al., 2008). While the queen throughout her life will feed on this substance (Page et al., 1998.)

Concerning to the protein composition of RJPs, it has been studied in the secretion of A. mellifera but, it is unknown in G. acapulconis. In A. mellifera had been described MRJP and YPs as RJPs, and some other proteins that bind MRJP to transport proteins or as defense proteins. Unfortunately, the RJP secretion in G. acapulconis implies to destroy the hives, so transcriptomic analysis of jelly-producing glands could provide indirect insight into the protein composition of RJPs in G. acapulconis.

Regarding the transcriptome of HG in bees, to our knowledge only they have been studied in A. mellifera. Therefore, the present communication improves and expand such data for A. mellifera, and for the first time, in G. acapulconis by high-throughput RNA sequencing.

2.1 Biological samples and RNA extraction

To identify the HG in A. mellifera and G. acapulconis, a head dissection was performed using a scalpel, tweezers, and sterile scissors, using a stereoscopic microscope. The heads of G. acapulconis and A. mellifera species were dissected and the HG separated for RNA extraction (Fig. S1). The tissues were placed in TRIzol® and stored at -20°C until use. The organs were macerated in a previously sterilized homogenizer. RNA was extracted using the TRIzol® reagent, guanidine/phenol isothiocyanate method (Chomczynski & Sacchi, 1987). At the end, the pellet obtained was dissolved in 50 µL of H₂O/Dpc. All reagents used were RNase-free, and the material used was sterile. We dissected thirty bees for each specie, due to the dimensions of the bees, it was decided to cut the head, eliminating the antennae and the tongue. Then, the HG were dissected, and the extracted material was stored in TRIzol®.

2.2 cDNA library preparation and Illumina sequencing

A double-stranded cDNA library was constructed from the obtained total RNA, using the high-quality TruSeq® library preparation kits. The library was sequenced in NextSeq® 500 System WGS Solution with the standard protocol. An automated sequencing was performed at University Unit for Massive Sequencing and Bioinformatics (UUSMB) of the Institute of Biotechnology, of the National Autonomous University of Mexico (IBt-UNAM).

2.3 Assembly, annotation and Bioinformatic analysis

After adaptor clipping, we removed low-quality sequences with programs cutadapt 1.18 and trimmomatic 0.39. The de novo assembly was performed using Trinity v. 3.4., with standard protocol. The prediction and translation of coding regions was performed with the TransDecoder program. The annotation was performed with Trinotate v3.1.1, CD-HIT 18.04 and Tblastn 2.12.0. Trinotate annotation includes homology searches with known sequence databases (BLAST+/SwissProt/Uniref90), protein domain identification (HMMER/PFAM), prediction of signal peptide and transmembrane domains (signalP/tmHMM), and comparison with currently curated annotation databases (BLAST+/SwissProt/Uniref90; EMBL Uniprot eggNOG/GO Pathways) (Tellez-Garcia et al., 2021). In addition, we performed annotation protein sequence with CD-HIT and a homolog search with Tblastn using a specialized database of RJPs obtained from UniProt (https://www.uniprot.org/05/17/2022). In the case of tblastn, only transcripts with a minimum e-value of 1e-4, 70% homology and a minimum size of 200 amino acids were recovered. Finally, for high-quality functional annotation and analysis of genomic datasets were determined using Blast2GO (https://www.blast2go.com/).

3.1 Sample Collection

Specimens of G. acapulconis were collected in Xoxocotla, Morelos. For A. mellifera, they were collected in the apiary of the Autonomous University of the State of Morelos (UAEM) in Cuernavaca, Morelos. G. acapulconis was identified by Dr. Ricardo Ayala Barajas from the Institute of Biology belonging to the National Autonomous University of Mexico (UNAM).

Thirty individuals were randomly collected for each species. In addition to the most notable differences in size (~ 3 mm in G. acapulconis and ~ 20 mm in A. mellifera), color differences were observed (Fig. 1).

Apart from the size, the differences observed in the figure are the pigment of the body and the number of hairs they contain as well as the color itself. G. acapulconis is completely black from head to abdomen with almost no noticeable white hairs on the sides and abdomen. A. mellifera is orange containing a black spot on the body and black lines on the abdomen, with yellow hairs on the thorax and no yellow hairs on the abdomen.

3.2 Dissection and extraction

We obtained a total RNA concentration of 308.0 and 157.7 ng/µL for A. mellifera and for G. acapulconis, respectively. The RNA samples were evaluated in an Agilent 2100 Bioanalyzer chip. The RNA results presented a signal clearly corresponding to the 18S ribosomal RNA, but a very low signal to the 28S. Like other arthropod samples, it was not possible to determine the RIN (RNA Integrity Number) due to a fracture (disruption) in the 28S ribosomal, resulting in two fragments of the same size as the 18S ribosomal. This phenomenon has also been observed during the analysis of Apis mellifera RNA (Winnebeck et al., 2010).

3.3 Quality analysis and assembly.

After Illumina sequencing, we obtained 11,684,728 and 13,209,338 reads for G. acapulconis and A. mellifera, respectively, from which cleaning of libraries were performed resulting in 10,492,794 and 12,585,393 reads for G. acapulconis and A. mellifera, respectively. Subsequently, we performed de novo assemblies with Trinity, from which 68,721 putative transcripts for A. mellifera and 88,510 for G. acapulconis were obtained. Afterwards, we performed the annotation with Trinotate, CD-hit and Tblastn (Table 1). We observed a higher number of annotated transcripts (33,339) in G. acapulconis with Trinotate and CD-hit compared to those annotated transcripts (28,099) found in A. mellifera.

Table 1

Transcripts annotated in Trinotate, CD-HIT and Tblastn
	G. acapulconis
Analysis	Trinotate	CD-HIT	Tblastn	Trinotate	CD-HIT	Tblastn
Transcripts	33,339	15,521	214	28,099	12,843	524
SprotTopBLASTPhit	17,898	14,473		14,816	12,020
COG	1,404	4,300		1,832	3,408
Pfam	15,820	13,018		13,406	11,081
Gene Ontology Blast	4,880			4,915

It is noticeable, that despite the higher annotation rate from Trinotate for G. acapulconis more COG and GO assignments were found for A. mellifera. We used COG annotated sequences to generally compare both transcriptional profiles (Fig. 2), which shows that it is not evident differences between them.

On the other hand, this comparation provide a peak of which category are more represent in bees. We found that J, K, O, S, T and U protein groups had a great diversity of transcripts. Remarkably, the G category include proteins for carbohydrate metabolism and transport, since in that classification Yellow/MRJP and RJPL proteins are found, we observed similar proportions in this category for both bees. It should be noted that, despite the difference between the number of transcripts between the assemblies, the profiles classified by COG do not present obvious differences in diversity of the transcripts.

Due to the approach of this work, it is known and understood the possible content of RJPs secreted by HG in both bees. We obtained transcripts identified as Yellow/MRJP and RJPL proteins by CD-HIT, Trinotate and TBLASTN. These protein sequences were also taken to recover missing information and to reaffirm the data already obtained. After this process, we obtained 105 and 22 proteins for A. mellifera and G. acapulconis, respectively. As described in literature A. mellifera had transcripts coding MRJP1 to MRJP9 and YPs. In G. acapulconis, it was found YPs, RJPL and Vitellogenin (MRJP1VIT). MRJP1VIT is a protein that bind to MRJP1 and allow to such protein to carry out its biological function. Despite both bees had YPs, we observed that G. acapulconis has a higher number of yellow transcripts than A. mellifera with 75% and 9%, respectively (Fig. 3).

3.4 Blast2GO

To better understand the function of differentially expressed genes, we conducted an enrichment analysis for the Royal Jelly transcripts, the local database used in the Blast2go program was extracted from Uniprot (https://www.uniprot.org/05/17/2022), which yielded the following three groups: 1) Biological process, 2) Molecular function, and 3) Cellular component.

Fifty-two sequences were assigned in the category of biological process grouping them into 16 different GOs (Fig. 4). Most of them are from metabolic process as lipid metabolism, but other GO terms were related to response to stress, stimulus, and defense (Table S1).

Regarding molecular function transcripts (Fig. 5), we found 26 sequences in this category, which were grouped into 7 different GOs. The GO terms obtained were related to transport and binding proteins.

Finally, 90 sequences were assigned as cellular component grouping into 3 different GOs (Fig. 6).

3.5 Sequencing

The transcriptome raw reads were deposited in the Sequence Read Archive (SRA) bioproject number PRJNA943259 under the accession numbers to SUB12970917 and SUB12948074 for G. acapulconis and A. mellifera, respectively. The full database of contigs and protein sequences was uploaded to the Transcriptome Shotgun Assembly (TSA) under accession numbers GKLR01 (Meli_trinity_trim.fsa) and GKLQ01 (Apis_trinity_trim.fsa) for G. acapulconis and A. mellifera, respectively. The amino acid sequences of RJP, MRJP and YP proteins are found in Table S2.

Through the manuscript, we mentioned several morphological differences between A. mellifera and G. acapulconis. We suggest that the content of RJPs is related to those morphological differences. Because some of those proteins that have been described are involved into pigmentation and larval development. We used a transcriptomic approach by HG, which produce RJPs to identify their coding sequences. Transcriptomic analysis shows that the transcription profiles for A. mellifera and G. acapulconis are similar, but the differences are in the diversity from RJPs.

Regarding MRJP family, it has been described MRJP1 to MRP10, which are related to different activities (Ferguson et al., 2011). MRJP1-5 have nutritional and defense function while, MRJP6, MRJP7 and MRJP10 activity remain unknown (Schmitzová et al., 1998). On another hand, MRJP8 and MRJP9 are related to honeybee venom (Blank et al., 2012; Peiren et al., 2008). We found that A. mellifera had coding sequences to MRJPs and YPs. On the contrary, G. acapulconis showed a higher diversity to YPs, and MRJP were absent, instead those proteins found were RJPL. In relation to MRJP family, we found transcripts coding for MRJP1 to MRJP5, MRJP8 and MRJP9 in A. mellifera.

Concerning MRJP1, it is expressed in HG and brain (Hojo et al., 2010; Kucharski et al., 1998; Peixoto et al., 2009). MRJP1 is related to different activities such as a growth factor in the worker/queen transition (Kamakura, 2011). MRJP1 is closely related to apisimin, a 54-residue alpha-helical peptide that promotes the non-covalent assembly of MRJP1 into multimers (Bíliková et al., 2002, 2009). High-resolution structural data for these complexes are not available, and their binding stoichiometry remains uncertain. To date, MRJP1/Apisimin has been studied using a range of biophysical techniques (Mandacaru et al., 2017). Also, the expression of this protein in the brain has been reported in mushroom bodies (MBs) or in stalk-shaped Kenyon cells, which could indicate a non-nutritive function. MBs have been shown to be involved in sensory integration and learning and memory in bees (Hourcade et al., 2010; Komischke et al., 2005; Menzel, 2001). One possibility could be that they have function as a growth factor involved in Kenyon cells growth or plasticity (Kucharski et al., 1998).

Also, a single-copy of MRJP-like has been found in bumblebees, which may represent the ancestral state of MRJP evolution (Kupke et al., 2012).

There is evidence that MRJP2 protect larvae from infections because they are homologs of apalbumins which are antimicrobial peptides (Bíliková et al., 2002, 2009). In a similar way, MRJP3 could be act as defence protein, because they had influence in T-cell immune response by regulating the production of IL-4, IL- 2 and IFN-γ (Kohno et al., 2004). They also report that MRJP3 inhibits IgE and IgG1 production and effectively acts as an anti-allergy agent. Additionally, MRJP3 is known to act as an anti-inflammatory agent both in vitro and in vivo in macrophages. The presence of MRJP2 and MRJP3 in A. mellifera could be related to differences in bees’ development that we mentioned before. After the workers build the brood cells, G. acapulconis provide them with larval food and close the brood, while the A. mellifera keep the brood cell open, so that the workers can feed the brood from time to time. This developmental difference makes A. mellifera more susceptible to diseases. For that reason, MRJP2 and MRJP3 could play as defense proteins, and so far, what is known about MRJP4 and MRJP5 is that they contain nutritional function.

As we mentioned before, in G. acapulconis the MRJP were absent, but instead those proteins we found RJPL. This protein has been reported in bumblebees (Albert et al., 2014; Kupke et al., 2012; Terzo et al., 2007). It has been described that RJPL expression occurs in both HGs and Kenyon cells of the brain, suggesting that this type of expression pattern is not bee-specific, but may represent an ancestral rather than a derived state (Albert et al., 2014). In relation to YPs, they were found in both bees. However, the diversity of this kind of proteins was higher in G. acapulconis. YPs have multiples activities, they have been associated with development, regulatory functions, and behavior of bees (Buttstedt et al., 2014; Drapeau et al., 2006; Ferguson et al., 2011). Specifically, YPs are the oldest and best characterized proteins in the Yellow/MRJP family. Since MRJP and YP are multifunctional, the exact biological function of several MRJPs remain unknown; so, we do not know the repercussions for the bee species. For example, several Yellow/MRJP family have the intrinsic ability to bind and modify biogenic amines such as DOPA and dopamine (Qian HAN et al., 2002; Xu et al., 2011); therefore, they affect neuromodulation and dopaminergic transmission, which it is common in the insect brain (Blenau & Erber, 1998). It is possible that neuronal MRJPs are directly or indirectly involved in this mode of neuronal communication (Albert et al., 2014). Also, YPs have been found in other insects as Drosophila melanogaster, in which is synthesized in the brain. Further, this protein plays a critical role for male courtship behavior modulation (Radovic et al., 2002), but also is related to fly pigmentation. Therefore, the diversity of YPs in G. acapulconis could be explain the pigmentation differences. However, since other YPs had been described to have multiples activities, some of them have similar MRJP activities, suggesting that the YPs in G. acapulconis replaced MRJP.

In G. acapulconis, it may has greater YP diversity than A. mellifera to reinforce the remaining MRJP proteins of G. acapulconis. If MRJP proteins are absent in G. acapulconis a higher amount of RJPL would be expected. The predominant proteins found in G. acapulconis were YP and RJPL. However, G. acapulconis does have proteins that react with MRJP1, which is MRJP1VIT, it could be that RJPL, or some YPs play a similar role as MRJP1.

Finally, in both bees we found Vitellogenin (MRJP1VIT) a protein that has a close relationship to MRJP proteins because they bind to MRJP1 proteins to facilitate the transport.

In this study, a comparison of the transcriptome from the HGs from G. acapulconis and from A. mellifera was obtained by high-throughput sequencing. This work reinforces the importance of understating the biological role of the HGs from G. acapulconis, contributes to the knowledge of its composition and may assists to the comprehension of the RJPs from this specie.

Acknowledgements

The research work was funded by DGAPA-UNAM (grant number IT200321) and by CONACyT (grant number 303045).

Supplementary Materials

Figure S1. Glandular system in the head of A. mellifera.

Table S1. GOs obtained with the database.

Table S2. The amino acid sequences of RJP, MRJP and YP proteins

Author Contributions: Conceptualization: JC, GC and EC; methodology and analysis: EC, GP, HC and JC; Writing: EC, GC and JC, review and editing: GC and JC; funding acquisition: GC.

Conflicts of Interest: The authors declare no conflict of interest.

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TableS2.xlsx
Table S2. The amino acid sequences of RJP, MRJP and YP proteins

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Transcriptomic comparison of the royal jelly proteins coded in the hypopharyngeal glands of Apis mellifera and Geotrigona acapulconis

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Biological samples and RNA extraction

2.2 cDNA library preparation and Illumina sequencing

2.3 Assembly, annotation and Bioinformatic analysis

3. Results

3.1 Sample Collection

3.2 Dissection and extraction

3.3 Quality analysis and assembly.

3.4 Blast2GO

3.5 Sequencing

Discussion

Conclusion

Declarations

References

Supplementary Files

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