Honey Bee Caste Research Articles

Identification of low-abundance proteins in the royal jelly using the Osborne classification method

Royal jelly (RJ) is recognized as healthy food, with a high content of proteins. These proteins play important roles in honeybee caste and human health, but the proteomic analysis of low-abundance proteins in RJ has long been a challenge. Herein, we used the Osborne classification method to separate the RJ proteins of Xinjiang black bees into various fractions. The globulin, ethanol-soluble protein, and glutelin fractions were further separated by SDS-PAGE, and proteomic analysis was carried out by LC-MS/MS and searched against the UniProt database. A total of 23 secretory proteins were identified by proteomic analysis, in which 7 proteins were identified for the first time in RJ. The Osborne classification method combining one-dimensional gel electrophoresis-based proteomic analysis allows the identification of low-abundance proteins in the RJ and greatly extends the knowledge about the components and functions of RJ proteins. The raw data are available via ProteomeXchange with the identifier PXD023315. SignificanceThis study makes an important contribution to the research of the components and functions of low-abundance royal jelly proteins for the following reasons:1.The Osborne method was applied to fractionize the royal jelly proteins, which benefits the identification of low-abundance proteins.2.23 secretory proteins were identified from the globulins, the ethanol-soluble proteins, and the glutelins of the RJ, and there were 7 proteins identified for the first time in royal jelly.3.This study extends the knowledge about the components and functions of royal jelly proteins.

Dynamic Evolution of Repetitive Elements and Chromatin States in Apis mellifera Subspecies.

In this study, we elucidate the contribution of repetitive DNA sequences to the establishment of social structures in honeybees (Apis mellifera). Despite recent advancements in understanding the molecular mechanisms underlying the formation of honeybee castes, primarily associated with Notch signaling, the comprehensive identification of specific genomic cis-regulatory sequences remains elusive. Our objective is to characterize the repetitive landscape within the genomes of two honeybee subspecies, namely A. m. mellifera and A. m. ligustica. An observed recent burst of repeats in A. m. mellifera highlights a notable distinction between the two subspecies. After that, we transitioned to identifying differentially expressed DNA elements that may function as cis-regulatory elements. Nevertheless, the expression of these sequences showed minimal disparity in the transcriptome during caste differentiation, a pivotal process in honeybee eusocial organization. Despite this, chromatin segmentation, facilitated by ATAC-seq, ChIP-seq, and RNA-seq data, revealed a distinct chromatin state associated with repeats. Lastly, an analysis of sequence divergence among elements indicates successive changes in repeat states, correlating with their respective time of origin. Collectively, these findings propose a potential role of repeats in acquiring novel regulatory functions.

The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees.

Honey bees are typical model organisms for the study of caste differentiation, and the juvenile hormone (JH) is a crucial link in the regulatory network of caste differentiation in honey bees. To investigate the mechanism of JH-mediated caste differentiation, we analyzed the effect of the JH response gene AmKr-h1 on this process. We observed that AmKr-h1 expression levels were significantly higher in queen larvae than in worker larvae at the 48 h, 84 h, and 120 h larval stages, and were regulated by JH. Inhibiting AmKr-h1 expression in honey bee larvae using RNAi could lead to the development of larvae toward workers. We also analyzed the transcriptome changes in honey bee larvae after AmKr-h1 RNAi and identified 191 differentially expressed genes (DEGs) and 682 differentially expressed alternative splicing events (DEASEs); of these, many were related to honey bee caste differentiation. Our results indicate that AmKr-h1 regulates caste differentiation in honey bees by acting as a JH-responsive gene.

Effects of seasonal variations on the development of different castes of honeybees (Apis mellifera)

Effects of seasonal variations on the development of different castes of honeybees (Apis mellifera)

Respiration levels of honey bee castes as a possible indicator in Varroa mite attraction

It has been demonstrated that fatty acids esters play an important role in the caste selection Varroa mites. However, there may be another mechanism of attraction in the preferred host location. We hypothesize that it may be due to differential respiration rates between larvae, but no investigations have been reported. This research was developed to determine if there is a measurable level and significant difference in honey bee caste respiration that may be exploited by Varroa mites in choice selection. In this study, we measured the respiration of all stages of the Western honey bee Apis mellifera, L. castes to determine the rate of continuous respiration. An assay was conducted using a portable photosynthesis system for gas exchange and chlorophyll fluorescence measurement (Li-Core LI-6400XT) to measure the respiration rate. In this study, a comparative analysis resulted in respiration levels that indicated a significant difference in the CO2 levels of adult worker (777.0 ± 0.002 μg CO2 g insect-1 min-1) and drone (375.5 µg CO2) honey bees. The comparison of worker (5.59 ± 0.002 μg CO2 g insect-1 min-1) and drone (7.20 ± 0.002 μg CO2 g insect-1 min-1) pupae showed no significant difference in the respiration levels; however, there was a noticeable difference in respiration levels of the worker (6.33 ± 0.002 μg CO2 g insect-1 min-1) and drone (10.07 ± 0.002 μg CO2 g insect-1 min-1) larvae. Future research will utilize the knowledge obtained to examine the mite’s attraction to the honey bee castes in host location. This information is a crucial step for the advancement of research studies and the development of management protocols for Varroa.

Expression of subunits of an insecticide target receptor varies across tissues, life stages, castes, and species of social bees.

Global losses of insects jeopardize ecosystem stability and crop pollination. Robust evidence indicates that insecticides have contributed to these losses. Notably, insecticides targeting nicotinic acetylcholine receptors (nAChRs) have neurotoxic effects on beneficial insects. Because each nAChR consists of five subunits, the alternative arrangements of subunits could create a multitude of receptors differing in structure and function. Therefore, understanding whether the use of subunits varies is essential for evaluating and predicting the effects of insecticides targeting such receptors. To better understand how the use and composition of nAChRs differ within and between insect pollinators, we analysed RNA-seq gene expression data from tissues and castes of Apis mellifera honey bees and life stages and castes of the Bombus terrestris bumble bees. We reveal that all analysed tissues express nAChRs and that relative expression levels of nAChR subunits vary widely across almost all comparisons. Our work thus shows fine-tuned spatial and temporal expression of nAChRs. Given that coexpression of subunits underpins the compositional diversity of functional receptors and that the affinities of insecticides depend on nAChR composition, our findings provide a likely mechanism for the various damaging effects of nAChR-targeting insecticides on insects. Furthermore, our results indicate that the appraisal of insecticide risks should carefully consider variation in molecular targets.

Detection and pH-Thermal Characterization of Proteinases Exclusive of Honeybee Worker-Fate Larvae (Apis mellifera L.)

The occurrence of the honeybee caste polyphenism arises when a change in diet is transduced into cellular metabolic responses, resulting in a developmental shift mediated by gene expression. The aim of this investigation was to detect and describe the expression profile of water-soluble proteases during the ontogenesis of honeybee worker-fate larvae. The extraction of insect homogenates was followed by the electrophoretic separation of the protein extract in polyacrylamide gels under semi-denaturing condition, precast with gelatin, pollen, or royal jelly protein extracts. The worker-fate honeybee larva showed a proteolytic pattern that varied with aging, and a protease with the highest activity at 72 h after hatching was named PS4. PS4 has a molecular weight of 45 kDa, it remained active until cell sealing, and its enzymatic properties suggest a serine-proteinase nature. To define the process that originates a queen-fate larvae, royal jelly and pollen were analysed, but PS4 was not detected in either of them. The effect of food on the PS4 was investigated by mixing crude extracts of queen and worker-fate larvae with pollen and royal jelly, respectively. Only royal jelly inhibited PS4 in worker-fate larvae. Taken together, our data suggest that PS4 could be involved in caste differentiation.

Sex-Biased Expression of Olfaction-Related Genes in the Antennae of Apis cerana (Hymenoptera: Apidae).

The olfactory system is essential for honeybees to adapt to complex and ever-changing environments and maintain cohesiveness. The Eastern honeybee Apis cerana is native to Asia and has a long history of managed beekeeping in China. In this study, we analysed the antennal transcriptomes of A. cerana workers and drones using Illumina sequencing. A total of 5262 differentially expressed genes (DEGs) (fold change > 2) were identified between these two castes, with 2359 upregulated and 2903 downregulated in drones compared with workers. We identified 242 candidate olfaction-related genes, including 15 odourant-binding proteins (OBPs), 5 chemosensory proteins (CSPs), 110 odourant receptors (ORs), 9 gustatory receptors (GRs), 8 ionotropic receptors (IRs), 2 sensory neuron membrane proteins (SNMPs) and 93 putative odourant-degrading enzymes (ODEs). More olfaction-related genes have worker-biased expression than drone-biased expression, with 26 genes being highly expressed in workers’ antennae and only 8 genes being highly expressed in drones’ antennae (FPKM > 30). Using real-time quantitative PCR (RT-qPCR), we verified the reliability of differential genes inferred by transcriptomics and compared the expression profiles of 6 ORs (AcOR10, AcOR11, AcOR13, AcOR18, AcOR79 and AcOR170) between workers and drones. These ORs were expressed at significantly higher levels in the antennae than in other tissues (p < 0.01). There were clear variations in the expression levels of all 6 ORs between differently aged workers and drones. The relative expression levels of AcOR10, AcOR11, AcOR13, AcOR18 and AcOR79 reached a high peak in 15-day-old drones. These results will contribute to future research on the olfaction mechanism of A. cerana and will help to better reveal the odourant reception variations between different biological castes of honeybees.

Lipidomic specializations of honeybee (Apis mellifera) castes and ethotypes

Honeybees of the same colony combine a near-homogeneous genetic background with a high level of phenotypic plasticity, making them ideal models for functional lipidomics. The only external lipid source of the colony is pollen, a diet rich in polyunsaturated fatty acids (PUFA). It has been suggested that differences in exposure to pollen-derived PUFA could partly explain differences in longevity between honeybee castes. We here investigated whether the membrane composition of honeybees plays roles in the physiological adaptation to tasks of individuals within the colony. Membranes of cell heaters, a group of workers producing heat from their flight muscles to uphold brood nest temperature, were compared to those of different types of non-heaters. We found that the lipidomic profiles of these groups fall into clearly different “lipotypes”, characterized by chain length and saturation of phospholipid-bound fatty acyl residues. The nutritional exposure to PUFA during early adult life and pupal development at the lower edge of the natural range of brood nest temperature both suppressed the expression of the cell heater-“lipotype”. Because cardiolipins (CL) are the lipid class most clearly differentiating honeybee phenotypes, and CL plays central roles in mitochondrial function, dysfunction and aging, our findings could help to understand these processes in other animals and humans.Taken together, the lipidome analysis of different life stages of workers, fertile queens, and drones lead to the hypothesis that honeybee “lipotypes” might represent adaptations to different energetic profiles and the likelihood of exposure to low temperatures.

The role of honey in the ecology of the hive: Nutrition, detoxification, longevity, and protection against hive pathogens.

Honey is the source of energy for the European honey bee, Apis mellifera. Beyond simple nutrition and a hedge against the seasonal, geographic, and chemical unpredictability of nectar, honey has properties that protect the hive against various stresses. Enzyme-mediated detoxification during honey ripening neutralizes potentially toxic phytochemicals, and bees that consume honey have enhanced tolerance to other ingested toxins. Catalase and antioxidant phenolics protect honey bees from oxidative damage caused by reactive oxygen species, promoting their longevity. Phytochemical components of honey and microRNAs have the potential to influence developmental pathways, with diet playing a large role in honey bee caste determination. Components of honey mediate stress response and promote cold tolerance during overwintering. Honey has a suite of antimicrobial mechanisms including osmotic pressure, low water activity, low pH, hydrogen peroxide, and plant-, honey bee-, and microbiota-derived compounds such as phytochemicals and antimicrobial peptides. Certain types of honey, particularly polyfloral honeys, have been shown to inhibit important honey bee pathogens including the bacteria responsible for American and European Foulbrood, the microsporidian Nosema ceranae, and the fungi responsible for Stonebrood. Understanding the diverse functional properties of honey has far-ranging implications for honey bee and hive health and management by beekeepers.

Larval Exposure to Parasitic Varroa destructor Mites Triggers Specific Immune Responses in Different Honey Bee Castes and Species.

Innate immune systems are key defenses of animals and particularly important in species that lack the sophisticated adaptive immune systems as found in vertebrates. Here, we were interested to quantify variation in innate immune responses of insects in hosts that differ in their parasite susceptibility. To do this, we studied immune responses in honey bees, which can host a remarkable number of different parasites, which are major contributors of declining bee health and colony losses. The most significant parasite of honey bees is the mite Varroa destructor, which has infested the majority of global honey bee populations, and its control remains a major challenge for beekeepers. However, a number of nonmanaged honey bees seem able to control Varroa infections, for example, the Eastern honey bee Apis ceranacerana or the African honey bee Apis mellifera scutellata. These bees therefore make interesting study subjects to identify underlaying resistance traits, for example, by comparing them to more susceptible bee genotypes such as Western honey bees (A. melliferaligustica). We conducted a series of interlinked experiments and started with behavioral assays to compare the attractiveness of bee larvae to mites using different honey bee genotypes and castes. We found that 6-day-old larvae are always most attractive to mites, independently of genotype or castes. In a next step, we compared volatile profiles of the most attractive larvae to test whether they could be used by mites for host selection. We found that the abundance of volatile compounds differed between larval ages, but we also found significant differences between genotypes and castes. To further study the expected underlaying physiological differences between potentially resistant and susceptible host larvae, we compared the larval hemolymph proteomes of the three honey bee genotypes and two castes in response to mite exposure. We identified consistent upregulation of immune and stress-related genes in Varroa-exposed larvae, which differed between genotypes and castes. Tolerant honey bee castes and genotypes were characterized by stronger or more distinct immune esponses. In summary, we provide first insights into the complex involvement of the innate immune system of tolerant honey bees against mite infestations, which could be used for future breeding purposes.

Juvenile hormone and transcriptional changes in honey bee worker larvae when exposed to sublethal concentrations of thiamethoxam

Thiamethoxam, an insecticide with high usage and large amounts of environmental residues, has been reported to affect the pupation and survival of honey bee larvae at sublethal concentrations. The molecular mechanisms are not fully understood. In this study, we measured the response of juvenile hormone (JH) to environmental concentrations of thiamethoxam using liquid chromatography-tandem mass spectrometry (LC-MS/MS), monitored the dynamic changes in the transcription of genes encoding major JH metabolic enzymes (CYP15A1, FAMET, JHAMT and JHE) using RT-qPCR, and analysed the transcriptome changes in worker larvae under thiamethoxam stress using RNA-seq. Thiamethoxam significantly increased the levels of JH3 in honey bee larvae, but no significant changes in the transcript levels of the four major metabolic enzymes were observed. Thiamethoxam exposure resulted in 140 differentially expressed genes (DEGs). P450 CYP6AS5 was upregulated, and some ion-related, odourant-related and gustatory receptors for sugar taste genes were altered significantly. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that amino acid metabolism and protein digestion and absorption were influenced by thiamethoxam. These changes may do harm to honey bee caste differentiation, foraging behaviour related to sensory perception and nutrient levels of bee colonies. These results represent the first assessment of the effects of thiamethoxam on JH in honey bee larvae and provides a new perspective and molecular basis for the study of JH regulation and thiamethoxam toxicity to honey bees.

MiRNA-34 and miRNA-210 target hexamerin genes enhancing their differential expression during early brain development of honeybee (Apis mellifera) castes.

During the honeybee larval stage, queens develop larger brains than workers, with morphological differentiation appearing at the fourth larval phase (L4), just after a boost in nutritional difference both prospective females experience. The molecular promoters of this caste-specific brain development are already ongoing in previous larval phases. Transcriptomic analyses revealed a set of differentially expressed genes in the L3 brains of queens and workers, which represents the early molecular response to differential feeding females receive during larval development. Three genes of this set, hex70b, hex70c and hex110, are more highly transcribed in the brain of workers than in queens. The microRNAs miR-34, miR-210 and miR-317 are in higher levels in the queens' brain at the same phase of larval development. Here, we tested the hypothesis that the brain of workers expresses higher levels of hexamerins than that of queens during key phases of larval development and that this differential hexamerin genes expression is further enhanced by the repressing activity of miR-34, miR-210 and miR-317. Our transcriptional analyses showed that hex70b, hex70c and hex110 genes are differentially expressed in the brain of L3 and L4 larval phases of honeybee queens and workers. In silico reconstructed miRNA-mRNA interaction networks were validated using luciferase assays, which showed miR-34 and miR-210 negatively regulate hex70b and hex110 genes by directly and redundantly binding their 3'UTR (untranslated region) sequences. Taken together, our results suggest that miR-34 and miR-210 act together promoting differential brain development in honeybee castes by downregulating the expression of the putative antineurogenic hexamerin genes hex70b and hex110.

Amsp3 may act upstream of Amdnmt3 in female caste differentiation in the honeybee (Apis mellifera).

In honey bees, the process of producing two female castes, including queens and workers, is nutritionally controlled by differential feeding royal jelly to newly emerged larvae. Although they have almost identical genetic blueprints, these castes show striking differences in their morphologies, longevities and reproductive capabilities. DNA methyltransferase 3 (Amdnmt3) gene is involved in the regulatory network for honeybee caste differentiation. Due to the role of two zinc fingers containing transcription factors, SP1 and SP3 in controlling mammalian Dnmts, this study aimed to determine a similar interaction of SPs with Amdnmt3 in the honeybee. We confirmed that the promoter region of Amdnmt3 contained multiple predicted SP1/SP3 binding sites and then investigated the role of AmSP3 in queen-worker differentiation network. We observed that the expression level of Amsp3 was significantly higher in worker larvae than that in queen larvae at 48 h, 84 h and 120 h. Knockdown of Amsp3 expression by RNAi in worker larvae significantly reduced the expression level of Amdnmt3 and caused morphological changes in adult bees towards a queen-like phenotype. However, the expression levels of Amsp3 and Amdnmt3 were repressed by juvenile hormone (JH). Our results suggest that AmSP3 is an important part of the queen-worker differentiation network and supports the role of Amdnmt3 in determining the phenotypic outcome of developing larvae.

10-hydroxy-2E-decenoic acid (10HDA) does not promote caste differentiation in Melipona scutellaris stingless bees

In bees from genus Melipona, differential feeding is not enough to fully explain female polyphenism. In these bees, there is a hypothesis that in addition to the environmental component (food), a genetic component is also involved in caste differentiation. This mechanism has not yet been fully elucidated and may involve epigenetic and metabolic regulation. Here, we verified that the genes encoding histone deacetylases HDAC1 and HDAC4 and histone acetyltransferase KAT2A were expressed at all stages of Melipona scutellaris, with fluctuations between developmental stages and castes. In larvae, the HDAC genes showed the same profile of Juvenile Hormone titers—previous reported—whereas the HAT gene exhibited the opposite profile. We also investigated the larvae and larval food metabolomes, but we did not identify the putative queen-fate inducing compounds, geraniol and 10-hydroxy-2E-decenoic acid (10HDA). Finally, we demonstrated that the histone deacetylase inhibitor 10HDA—the major lipid component of royal jelly and hence a putative regulator of honeybee caste differentiation—was unable to promote differentiation in queens in Melipona scutellaris. Our results suggest that epigenetic and hormonal regulations may act synergistically to drive caste differentiation in Melipona and that 10HDA is not a caste-differentiation factor in Melipona scutellaris.

Caste-biased Expression of fem and Amdsx Genes in Apis mellifera ligustica (Hymenoptera: Apidae)

Sex determination and caste differentiation are two crucial processes for morphology building in honeybees. It is unclear whether there is an interaction between these two processes. Here, we investigated the expression of fem and Amdsx genes between female castes of honeybees. We found that the expression of fem and Amdsx is significantly higher in queens than in workers, and this expression was positively regulated by juvenile hormone (JH). Our results suggest that sex-determining genes fem and Amdsx are also involved in honeybee caste differentiation.

Dopamine production in the brain is associated with caste-specific morphology and behavior in an artificial intermediate honey bee caste.

Caste polymorphism in eusocial insects is based on morphological plasticity and linked to physiological and behavioral characteristics. To test the possibility that dopamine production in the brain is associated with the caste-specific morphology and behavior in female honey bees, an intermediate caste was produced via artificial rearing using different amounts of diet, before quantifying the dopamine levels and conducting behavioral tests. In field colonies, individual traits such as mandibular shape, number of ovarioles, diameter of spermatheca, and dopamine levels in the brain differed significantly between workers and queens. Females given 1.5 times the amount of artificial diet that control worker receives during the larval stage in the laboratory had characteristics intermediate between castes. The dopamine levels in the brain were positively correlated with the mandibular shape indexes, number of ovarioles, and spermatheca diameter among artificially reared females. The dopamine levels were significantly higher in females with mandibular notches than those without. In fighting experiments with the intermediate caste females, the winners had significantly higher dopamine levels in the brain than the losers. Brain levels of tyrosine were positively correlated with those of catecholamines but not phenolamines, thereby suggesting a strong metabolic relationship between tyrosine and dopamine. Thus, the caste-specific characteristics of the honey bee are potentially continuous in the same manner as those in primitively eusocial species. Dopamine production in the brain is associated with the continuous caste-specific morphology, as well as being linked to the amount of tyrosine taken from food, and it supports the aggressive behavior of queen-type females.

Caste and age-related changes in circulatory hemocytes of honey bee, Apis mellifera anatolica (Hymenoptera: Apidae)

In recent years, there has been a great concern about increased honey bee losses and this phenomenon has turned into a global issue. Therefore, immune system research on honey bees gained importance. Hemocytes are an important part of the immune system. But there is a limited study about hemocyte types of honey bees. From this point of view, a detailed study was performed on hemocyte types of honey bee Apis mellifera anatolica was described in larval (3rd, 4th and 5th larval instars), prepupal, pupal, and adult stages of all castes including worker, drone, and queen. According to light microscopic examinations, prohemocyte, plasmatocyte, granulocyte, adipohemocyte and oenocytoid were determined in all castes. Prohemocytes are usually rounded cells with a large nucleus and small cytoplasmic area. Plasmatocytes are spherical or oval cells. Granulocytes are generally spherical cells with dense granules. Adipohemocytes are usually spherical cells having numerous lipid droplets within their cytoplasm. Oenocytoides are usually spherical cells with small and spherical nucleus centrally located. Also, we statistically compared the size of hemocyte types among these castes during metamorphosis. According to our measurements, the queen generally had the biggest hemocytes in comparison to the developmental stages in other castes. While statistically significant differences in the sizes of hemocyte types were mainly detected among castes of the pupal stage, there were no statistically significant differences in hemocyte sizes among castes of adult honey bees. In view of these findings, it may be stated that hemocyte profiles of A. m. anatolica vary based on the developmental stages of castes.

Alcohol intoxication resistance and alcohol dehydrogenase levels differ between the honeybee castes

Various animal models are used in the study of alcoholism, with the honeybee (Apis mellifera L.) among them. Here, we tested the hypothesis that foragers show higher intoxication resistance to alcohol than nurses, an issue thus far not investigated. To this end, we measured the latency to full sedation when exposed to alcohol in foragers, nurses and reverted nurses. In addition, we measured alcohol dehydrogenase (ADH) levels in these worker castes. Caste status was confirmed by comparison of the size of their hypopharyngeal glands. We detected high intoxication resistance to alcohol and presence of ADH in foragers. In nurses, we detected significantly lower intoxication resistance to alcohol and no ADH. These between-caste differences cannot be explained by the age difference between castes as in reverted nurses, characterized by similar age to foragers, we detected an intermediate intoxication resistance to alcohol and no ADH. Our results suggest possible natural exposure to alcohol in different castes of workers. As such, we further develop the honeybee as a model in alcoholism-related research and open new research avenues.

Evidence of transfer of miRNAs from the diet to the blood still inconclusive.

MicroRNAs (miRNAs) are short, non-coding, single-strand RNA molecules that act as regulators of gene expression in plants and animals. In 2012, the first evidence was found that plant miRNAs could enter the bloodstream through the digestive tract. Since then, there has been an ongoing discussion about whether miRNAs from the diet are transferred to blood, accumulate in tissues, and regulate gene expression. Different research groups have tried to replicate these findings, using both plant and animal sources. Here, we review the evidence for and against the transfer of diet-derived miRNAs from plants, meat, milk and exosome and their assimilation and putative molecular regulation role in the consuming organism. Some groups using both miRNAs from plant and animal sources have claimed success, whereas others have not shown transfer. In spite of the biological barriers that may limit miRNA transference, several diet-derived miRNAs can transfer into the circulating system and targets genes for transcription regulation, which adds arguments that miRNAs can be absorbed from the diet and target specific genes by regulating their expression. However, many other studies show that cross-kingdom transfer of exogenous miRNAs appears to be insignificant and not biologically relevant. The main source of controversy in plant studies is the lack of reproducibility of the findings. For meat-derived miRNAs, studies concluded that the miRNAs can survive the cooking process; nevertheless, our evidence shows that the bovine miRNAs are not transferred to human bloodstream. The most important contributions and promising evidence in this controversial field is the transference of milk miRNAs in exosomes and the finding that plant miRNAs in beebread regulate honeybee caste development, and cause similar changes when fed to Drosophila. MiRNAs encapsulated in exosomes ensure their stability and resistance in the harsh conditions presented in milk, bloodstream, and gastrointestinaltract to reinforce the idea of transference. Regardless of the model organism, the idea of source of miRNAs, or the approach—bioinformatics or in vivo—the issue of transfer of miRNAs from the diet remains in doubt. Our understanding of the cross-kingdom talk of miRNAs needs more research to study the transfer of “xenomiRs” from different food sources to complement and expand what we know so far regarding the interspecies transfer of miRNAs.