Longitudinal Gene Expression Analysis in Honey Bees

A total of five genes were investigated in this study, namely: 1- vitellogenin (Vg), 2- major royal jelly protein 1 (mrjp1), 3- acetylcholine esterase 2 (AChE-2), 4- superoxide dismutase-like (Rsod) and 5- thioredoxin 1 (Trx-1). Sequences of the primers and their amplicon sizes are given in Table 1. Two-step reverse transcription quantitative PCR (RT-qPCR) using BioRad iTaq SYBER Green Supermix 2X was conducted on three biological and technical replicates per sample on five time points enabling a greater longitudinal analysis of gene regulation. cDNA was synthesized from RNA extractions using BioRad iScript Kit following the manufacturer’s protocol. Target genes were normalized against two housekeeping genes (GAPDH, RPS18) known for their stability in honey bee tissues^{38 (link),62 (link)}.Table 1

Target genes investigated in this study, housekeeping genes, primer sequences, amplicon size and NCBI accession numbers.

Gene	Description	F/R	bp	NCBI Accession
Target
AChE-2	Acetylcholinesterase-2	GACGCGAAGACCATATCCGT TCTGTGTCCTTGAAGTCCGC	140	NM_001040230.1
Mrjp1	Major royal jelly protein 1	TGACCAATGGCATGATAAG GACCACCATCACCGACCT	98	NM_001011579.1
Vg	Vitellogenin	AACGCTTTTACTGTTCGCGG TATGCACGTCCGACAGATCG	128	NM_001011578.1
Rsod	Superoxide dismutase-like	GGAGCAGTATCTGCAATGGGA CGCTACAAAACGTGGTGGTT	141	XM_006558333.2
Trx-1	Thioredoxin-1	AATGCACCGGCTCAAGAACA CATGCGACAAGGATTGCACC	138	XM_393603.7
Housekeeping
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase 2	TACCGCTTTCTGCCCTTCAA GCACCGAACTCAATGGAAGC	142	XM_393605.7
RPS18	40S ribosomal protein S18	AATTATTTGGTCGCTGGAATTG TAACGTCCAGCAGAATGTGGTA	238	XM_625101.6

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Alburaki M., Madella S, & Cook S.C. (2023). Non-optimal ambient temperatures aggravate insecticide toxicity and affect honey bees Apis mellifera L. gene regulation. Scientific Reports, 13, 3931.

Publication 2023

A genes Acetylcholine esterase Biological Cdna Dehydrogenase Gapdh Gene regulation Genes Honey Housekeeping genes Phosphate Primer Protein Reverse transcription Ribosomal protein Royal jelly Superoxide dismutase Thioredoxin 1 Vitellogenin

Corresponding Organization : Agricultural Research Service

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Variable analysis

independent variables

Time points (five time points)

dependent variables

Expression levels of the following genes: vitellogenin (Vg), major royal jelly protein 1 (mrjp1), acetylcholine esterase 2 (AChE-2), superoxide dismutase-like (Rsod), and thioredoxin 1 (Trx-1)

control variables

Two housekeeping genes (GAPDH, RPS18) used for normalization of target gene expression

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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