Sublethal exposure to thiamethoxam and pyraclostrobin affects the midgut and Malpighian tubules of the stingless bee Frieseomelitta varia (Hymenoptera: Apidae: Meliponini).

The stingless bee Frieseomelitta varia Lepeletier 1836 (Hymenoptera: Apidae) is an essential pollinator in natural and agricultural ecosystems in the Neotropical region. However, these bees may be exposed to pesticides during foraging, which can affect both individuals and their colonies. One example comes from the use of pyraclostrobin (a fungicide) and thiamethoxam (an insecticide) for pest control in pepper crops, which F. varia visits. This study aimed to evaluate the isolated and combined sublethal effects of thiamethoxam (TMX) (0.000543 ng a.i./µL) and pyraclostrobin (PYR) (1.5 ng i.a./µL) on the morphology of the midgut and Malpighian tubules of F. varia workers. Results showed that both pesticides, regardless of the exposure time (through feeding during 48 h or 96 h), disturbed the morphology of the analyzed organs. Specifically, F. varia exposed orally to sublethal concentrations of thiamethoxam and pyraclostrobin, either alone or in combination, exhibited a higher rate of damage to the midgut (e.g., vacuolization, apocrine secretion, and cellular elimination) compared to the bees in the control groups, both after 48 h and 96 h of exposure. In Malpighian tubules, vacuolation is the only damage present. As the observed morphological alterations likely compromise the excretion and absorption functions, exposure to pyraclostrobin and thiamethoxam may lead to disturbances at both the individual and colony levels. These results highlight the urgent need for a future reassessment of the safety of fungicides and insecticides regarding their potential effects on bee populations.

Thiamethoxam toxicity on the stingless bee Friesiomelitta varia: LC50, survival time, and enzymatic biomarkers assessment.

Bees play a crucial role as pollinating insects in both natural and cultivated areas. However, the use of pesticides, such as thiamethoxam, has been identified as a contributing factor compromising bee health. The current risk assessment primarily relies on the model species Apis mellifera, raising concerns about the applicability of these assessments to other bee groups, including stingless bees. In this study, we investigated the acute toxicity of thiamethoxam on the stingless bee Frieseomelitta varia by determining the average lethal concentration (LC50) and mean lethal time (LT50). Additionally, we evaluated the enzymatic profile of Acetylcholinesterase (AChE), Carboxylesterase-3 (CaE-3), and Glutathione S-Transferase (GST), in the heads and abdomens of F. varia after exposure to thiamethoxam (LC50/10). The LC50 of thiamethoxam was determined to be 0.68 ng ai/µL, and the LT50 values were 37 days for the control group, 25 days at LC50/10, and 27 days at LC50/100. The thiamethoxam significantly decreased the survival time of F. varia. Furthermore, the enzymatic profile exhibited differences in CaE3 activity within one day in the heads and ten days in the abdomen. GST activity showed differences in the abdomen after one and five days of thiamethoxam exposure. These findings suggests that the abdomen is more affected than the head after oral exposure to thiamethoxam. Our study provides evidence of the toxicity of thiamethoxam at both the cellular and organismal levels, reinforcing the need to include non-Apis species in pollinator risk assessments. and provide solid arguments for bee protection.

Exposure of the stingless bee Melipona scutellaris to imidacloprid, pyraclostrobin, and glyphosate, alone and in combination, impair its walking activity and fat body morphology and physiology.

The stingless bee Melipona scutellaris performs buzz pollination, effectively pollinating several wild plants and crops with economic relevance. However, most research has focused on honeybees, leaving a significant gap in studies concerning native species, particularly regarding the impacts of pesticide combinations on these pollinators. Thus, this study aimed to evaluate the sublethal effects of imidacloprid (IMD), pyraclostrobin (PYR), and glyphosate (GLY) on the behavior and fat body cell morphology and physiology of M. scutellaris. Foragers were orally exposed to the different pesticides alone and in combination for 48 h. Bees fed with contaminated solution walked less, moved slower, presented morphological changes in the fat body, including vacuolization, altered cell shape and nuclei morphology, and exhibited a higher count of altered oenocytes and trophocytes. In all exposed groups, alone and in combination, the number of cells expressing caspase-3 increased, but the TLR4 number of cells expressing decreased compared to the control groups. The intensity of HSP70 immunolabeling increased compared to the control groups. However, the intensity of the immunolabeling of HSP90 decreased in the IMD, GLY, and I + G (IMD + GLY) groups but increased in I + P-exposed bees (IMD + PYR). Alternatively, exposure to PYR and P + G (PYR + GLY) did not affect the immunolabeling intensity. Our findings demonstrate the hazardous effects and environmental consequences of isolated and combined pesticides on a vital neotropical pollinator. Understanding how pesticides impact the fat body can provide crucial insights into the overall health and survival of native bee populations, which can help develop more environmentally friendly approaches to agricultural practices.

Profiling the neuroproteomics of honeybee brain: A clue for understanding the role of neuropeptides in the modulation of aggressivity.

The aggressivity is modulated in honeybee brain through a series of actions in cascade mode, with the participation of the neuropeptides AmAST A (59-76) and AmTRP (254-262). The aggressivity of honeybees was stimulated by injecting both neuropeptides in the hemocoel of the worker honeybees, which were submitted to behavioral assays of aggression. The brain of stinger individuals were removed by dissection and submitted to proteomic analysis; shotgun proteomic approach of honeybee brain revealed that both neuropeptides activate a series of biochemical processes responsible by production of energy, neuronal plasticity and cell protection. In addition to this, AmTRP (254-262) elicited the expression of proteins related to the processing of the potential of action and lipid metabolism; meanwhile AmAST A (59-76) elicited the metabolism of steroids and Juvenile hormone-related metabolism, amongst others. Apparently, the most complex biochemical process seems to be the regulation of ATP production, which occurs at two levels: i) by a subgroup of proteins common to the three experimental groups, which are over-/under-regulated through glycolysis, pyruvate pathway, Krebbs cycle and oxidative phosphorylation; ii) by a subgroup of proteins unique to the each experimental group, which seems to be regulated through Protein-Protein Interactions, where the protein network regulated by AmTRP (254-262) seems to be more complex than the other two experimental groups. SIGNIFICANCE: Recently we reported the effect of the neuropeptides AmAST A (59-76) and AmTRP (254-262) in the modulation of the aggressive behavior of the worker honeybees. Up to now it is known that the simple presence of the allatostatin and tachykinin-related-peptide in bee brain, is enough for inducing the aggressive behavior. However, nothing was known about how these neuropeptides perform their action, inducing the aggressive behavior. The results of the present study elucidated some of the metabolic pathways that were activated or inhibited to support the complex defensive behavior, which includes the aggressivity. These results certainly will impact the behavioral research of honeybees, since we are paving the way for understanding the molecular base of regulation, of individual /nest defense of honeybees.

Landscape and land use affect composition and nutritional values of bees' food.

Bees are primary pollinators across various terrestrial biomes and rely heavily on floral resources for sustenance. The composition of landscapes can influence bee foraging behavior, while human activities can directly affect both the composition and nutritional value of bee food. We aimed to assess how landscape structure and land use practices can impact the composition and nutritional value of food sources for two generalist social bee species, Apis mellifera and Scaptotrigona postica. Food samples were collected from twenty-five colonies of A. mellifera and thirteen of S. postica to examine how food composition and nutritional value may vary based on the extent of human land use and the composition of landscapes surrounding beekeeping sites. The pollen composition and nutritional value of A. mellifera were influenced by both land use practices and landscape heterogeneity. The number of patches determined total sugar and lipid content. Landscape heterogeneity affected pollen composition in S. postica, primarily due to the number of patches, while total sugar was affected by landscape diversity. Pollen nutritional value in S. postica was linked to land use, mainly meadow and vegetation, which influenced total sugar and dry matter. S. postica showed a higher sensitivity to land use changes compared to A. mellifera, which was more affected by landscape heterogeneity. Assuring landscape heterogeneity by preserving remaining forest patches around apiaries and meliponaries is crucial. Thoughtful land use planning is essential to support beekeeping activities and ensure an adequate quantity and quality of bee food resources.

Exposure to sublethal concentrations of imidacloprid, pyraclostrobin, and glyphosate harm the behavior and fat body cells of the stingless bee Scaptotrigona postica.

Pesticide use in agriculture threatens non-target insects such as bees. Considering the ecological and economic relevance of native bees, such as Scaptotrigona postica, and the insufficient studies on the effects of pesticides on their behavior and physiology, improving the current knowledge on this issue is essential. Therefore, this study investigated the sublethal effects of imidacloprid, pyraclostrobin, and glyphosate on the behavior and fat body cells of S. postica. Pesticide ingestion decreased the walking distance and mean velocity of bees compared to the control and solvent control groups. The oenocytes of the control groups were spherical, with central nuclei containing decondensed chromatin, and the trophocytes presented irregular morphology, with cells varying in shape and the cytoplasm filled with vacuoles and granules. However, bees exposed to pesticides showed extensive cytoarchitectural disruption in the fat body, such as vacuolization and shape changes in oenocytes and altered nuclei morphology in trophocytes. Moreover, pesticide exposure increased the number of atypical oenocytes and altered trophocytes, except for the PYR group, which showed a lower number of atypical oenocytes. Caspase-positive labeling significantly increased in all exposed bee groups. Alternatively, TLR4 labeling was significantly decreased in the exposed groups compared to the control groups. There was a significant increase in HSP90 immunolabeling in all exposed groups compared to the control. These findings reinforce the importance of research on the sublethal effects of low pesticide concentrations on key neotropical pollinators and prove that these toxic substances can impair their detoxification and immune defense.

Ultrastructural Changes in the Midgut of Brazilian Native Stingless Bee Melipona scutellaris Exposed to Fungicide Pyraclostrobin.

Melipona scutellaris is a Brazilian stingless bee that is important for pollinating wild flora and agriculture crops. Fungicides have been widely used in agriculture, and floral residues can affect forager bees. The goal of our study was to evaluate the effects of sublethal concentrations of pyraclostrobin on the midgut ultrastructure of M. scutellaris forager workers. The bees were collected from three non-parental colonies and kept under laboratory conditions. The bees were orally exposed continuously for five days to pyraclostrobin in syrup at concentrations of 0.125 ng a.i./µL (FG1) and 0.005 ng a.i./µL (FG2). The control bees (CTL) were fed a no-fungicide sucrose solution, and the acetone solvent control bees (CAC) received a sucrose solution containing acetone. At the end of the exposure, the midguts were sampled, fixed in Karnovsky solution, and routinely processed for transmission electron microscopy. Ultrastructural analysis demonstrated that both the fungicide concentrations altered the midgut, such as cytoplasmic vacuolization (more intense in FG1), the presence of an atypical nuclear morphology, and slightly dilated mitochondrial cristae in the bees from the FG1 and FG2 groups (both more intense in FG1). Additionally, there was an alteration in the ultrastructure of the spherocrystals (FG1), which could be the result of cellular metabolism impairment and the excretion of toxic metabolites in the digestive cells as a response to fungicide exposure. The results indicate that ingested pyraclostrobin induced cytotoxic effects in the midgut of native stingless bees. These cellular ultrastructural responses of the midgut are a prelude to a reduced survival rate, as observed in previous studies.

Sensitivity of the Neotropical Solitary Bee Centris analis F. (Hymenoptera, Apidae) to the Reference Insecticide Dimethoate for Pesticide Risk Assessment.

Currently, only Apis mellifera is used in environmental regulation to evaluate the hazard of pesticides to pollinators. The low representativeness of pollinators and bee diversity in this approach may result in insufficient protection for the wild species. This scenario is intensified in tropical environments, where little is known about the effects of pesticides on solitary bees. We aimed to calculate the medium lethal dose (LD50) and medium lethal concentration (LC50) of the insecticide dimethoate in the Neotropical solitary bee Centris analis, a cavity-nesting, oil-collecting bee distributed from Brazil to Mexico. Males and females of C. analis were exposed orally to dimethoate for 48 h under laboratory conditions. Lethality was assessed every 24 h until 144 h after the beginning of the test. After the LD50 calculation, we compared the value with available LD50 values in the literature of other bee species using the species sensitivity distribution curve. In 48 h of exposure, males showed an LD50 value 1.33 times lower than females (32.78 and 43.84 ng active ingredient/bee, respectively). Centris analis was more sensitive to dimethoate than the model species A. mellifera and the solitary bee from temperate zones, Osmia lignaria. However, on a body weight basis, C. analis and A. mellifera had similar LD50 values. Ours is the first study that calculated an LD50 for a Neotropical solitary bee. Besides, the results are of crucial importance for a better understanding of the effects of pesticides on the tropical bee fauna and will help to improve the risk assessment of pesticides to bees under tropical conditions, giving attention to wild species, which are commonly neglected. Environ Toxicol Chem 2023;42:2758-2767. © 2023 SETAC.

Surrogate species in pesticide risk assessments: Toxicological data of three stingless bees species.

Discussions about environmental risk reassessment of pesticides have grown in the last decades, especially in tropical and subtropical regions since the diversity of bee species in these places is quite different. Stingless bees are highly affected by pesticides, and toxicity information is necessary to include them in the regulatory process of countries that hosts a diversity of these species. Therefore, the present study aimed to evaluate the Median Lethal Concentration (LC50), estimate the Median Lethal Dose (LD50) and compared the sensitivity of three species of stingless bees exposed to the commercial formulation of the neonicotinoid thiamethoxam (TMX). The LD50 was estimated based on the LC50 determined in the present study (LC50 = 0.329 ng a.i./µL for Tetragonisca angustula; 0.624 ng a.i./µL for Scaptotrigona postica, and 0.215 ng a.i./µL for Melipona scutellaris). Considering these data, toxicity endpoints were used to fit species sensitive distribution curves (SSD) and determine the sensitivity ratio. The results showed that all the stingless bees tested are more sensitive to TMX than the Apis mellifera, the model organism used in ecotoxicological tests. Regarding the oral LC50, the most susceptible and most tolerant species were M. scutellaris > T. angustula > S. postica > A. mellifera. Following the same evaluated pattern, for the LD50 (considering the weight of the bees - ng a.i./g bee), we have: M. scutellaris > S. postica > T. angustula > A. mellifera, and without the weight considered (ng a.i./bee): T. angustula > M. scutellaris > S. postica > A. mellifera. The different sensitivities among stingless bee species highlight the importance of inserting more than one surrogate species with a variety of sizes in research and protocol development. Additionally, the research suggests the need to investigate patterns regarding the influence of body mass on pesticide sensitivity among stingless bee species.

Fungivorous mites enhance the survivorship and development of stingless bees even when exposed to pesticides.

Stingless bees are the largest group of eusocial bees in the world. They play an essential role as crop pollinators and have been considered for inclusion in pesticide risk assessments (RAs). Beyond the mutualism involving stingless bee larvae and fungi, the fungivorous mite Proctotydaeus (Neotydeolus) alvearii proved to be interesting for studies of associations with stingless bees. Their presence is related to colony strength and health, showing a permanent-host-association level. Here, we tested whether the coexistence with P. (N.) alvearii affects stingless bee larvae survivorship and development, including when fed pesticide-dosed food. We chose dimethoate, the reference standard for toxicity tests, and thiamethoxam, widely used in neotropical crops and listed to be reassessed in RAs. Bees associated with the mites showed higher larval survivorship rates, even in the dosed ones, and revealed changes in the developmental time and body size. Our study represents the first approach to stingless bee responses to the coexistence of fungivorous mites inside brood cells, leading us to believe that these mites play a beneficial role in stingless bees, including when they are exposed to pesticides.

Harmful Effects of Pyraclostrobin on the Fat Body and Pericardial Cells of Foragers of Africanized Honey Bee.

Managed honey bees are daily exposed in agricultural settings or wild environments to multiple stressors. Currently, fungicide residues are increasingly present in bees' pollen and nectar and can harm colonies' production and survival. Therefore, our study aimed to evaluate the effects of the fungicide pyraclostrobin on the fat body and pericardial cells of Africanized honey bees. The foragers were divided into three experimental treatment groups and two controls: pyraclostrobin 0.125 ng/µL (FG1), 0.025 ng/µL (FG2), 0.005 ng/µL (FG3), untreated control (CTL), and acetone control (CAC). After five days of oral exposure (ad libitum), the bees were dissected and prepared for histopathological and morphometric analysis. The FG1-treated bees showed extensive cytoarchitecture changes in the fat body and pericardial cells, inducing cell death. Bees from the FG2 group showed disarranged oenocytes, peripheral vacuolization, and pyknotic nuclei of pericardial cells, but the cytoarchitecture was not compromised as observed in FG1. Additionally, immune system cells were observed through the fat body in the FG1 group. Bees exposed to FG3 demonstrated only oenocytes vacuolization. A significant decrease in the oenocyte's surface area for bees exposed to all pyraclostrobin concentrations was observed compared to the CTL and CAC groups. The bees from the FG1 and FG2 treatment groups presented a reduced surface area of pericardial cells compared to the controls and the FG3 group. This study highlighted the harmful effects of fungicide pyraclostrobin concentrations at the individual bee cellular level, potentially harming the colony level on continuous exposure.

Transcriptomic analysis of Malpighian tubules from the stingless bee Melipona scutellaris reveals thiamethoxam-induced damages.

The concern about pesticide exposure to neotropical bees has been increasing in the last few years, and knowledge gaps have been identified. Although stingless bees, (e.g.: Melipona scutellaris), are more diverse than honeybees and they stand out in the pollination of several valuable economical crops, toxicity assessments with stingless bees are still scarce. Nowadays new approaches in ecotoxicological studies, such as omic analysis, were pointed out as a strategy to reveal mechanisms of how bees deal with these stressors. To date, no molecular techniques have been applied for the evaluation of target and/or non-target organs in stingless bees, such as the Malpighian tubules (Mt). Therefore, in the present study, we evaluated the differentially expressed genes (DEGs) in the Mt of M. scutellaris after one and eight days of exposure to LC50/100 (0.000543 ng a.i./µL) of thiamethoxam (TMX). Through functional annotation analysis of four transcriptome libraries, the time course line approach revealed 237 DEGs (nine clusters) associated with carbon/energy metabolism and cellular processes (lysosomes, autophagy, and glycan degradation). The expression profiles of Mt were altered by TMX in processes, such as detoxification, excretion, tissue regeneration, oxidative stress, apoptosis, and DNA repair. Transcriptome analysis showed that cell metabolism in Mt was mainly affected after 8 days of exposure. Nine genes were selected from different clusters and validated by RT-qPCR. According to our findings, TMX promotes several types of damage in Mt cells at the molecular level. Therefore, interference of different cellular processes directly affects the health of M. scutellaris by compromising the function of Mt.

Monitoring the effects of field exposure of acetamiprid to honey bee colonies in Eucalyptus monoculture plantations.

Eucalyptus plantations occupy 26 % of Portuguese forested areas. Its flowers constitute important sources for bees and beekeepers take advantage of this and keep their honey bee colonies within or near the plantations for honey production. Nonetheless, these plantations are susceptible to pests, such as the eucalyptus weevil Gonipterus platensis. To control this weevil, some plantations must be treated with pesticides, which might harm non-target organisms. This study aimed to perform a multifactorial assessment of the health status and development of Apis mellifera iberiensis colonies in two similar landscape windows dominated by Eucalyptus globulus plantations - one used as control and the other with insecticide treatment. In each of the two selected areas, an apiary with five hives was installed and monitored before and after a single application of the insecticide acetamiprid (40 g a.i./ha). Colony health and development, resources use, and pesticide residues accumulation were measured. The results showed that the application of acetamiprid in this area did not alter the health status and development of the colonies. This can be explained by the low levels of residues of acetamiprid detected only in pollen and bee bread samples, ~52 fold lower than the sublethal effect threshold. This could be attributed to the low offer of resources during and after the application event and within the application area, with the consequent foraging outside the sprayed area during that period. Since exposure to pesticides in such complex landscapes seems to be dependent on the spatial and temporal distribution of resources, we highlight some key monitoring parameters and tools that are able to provide reliable information on colony development and use of resources. These tools can be easily applied and can provide a better decision-taking of pesticide application in intensive production systems to decrease the risk of exposure for honey bees.

Optimization of in vitro culture of honeybee nervous tissue for pesticide risk assessment.

The most used pesticides have neurotoxic action on the neurotransmitter system of target and non-targeted insects, such as honeybees. However, honeybees have foremost importance worldwide, which has encouraged the development of tools to evaluate the action of specific pesticide molecules on their nervous system, providing accurate data on damage to their brain. In this sense, our study aimed to optimize in vitro honeybee nervous tissue culture to assess pesticide risks. To this end, six forager honeybee brains were dissected and transferred to different combinations of Leibovitz-15 (L-15) culture medium supplemented with Fetal Bovine Serum (FBS), Hank's Balanced Salt Solution (HBSS), and Insect Medium Supplement (IMS). Nervous tissues were collected after different incubation times (1, 6, 12, and 24 h) for morphology and Kenyon cell analyses. Our results showed that L-15 medium supplemented with HBSS and with HBSS plus FBS were the best media for culturing honey nervous tissue for 24 h, as they resulted in less tissue spacing and cell disarrangement. Therefore, they may be assessed in future ecotoxicological tests.

A food-ingested sublethal concentration of thiamethoxam has harmful effects on the stingless bee Melipona scutellaris.

In recent years, the importance of bee's biodiversity in the Neotropical region has been evidencing the relevance of including native bees in risk assessments. Therefore, the sublethal effects of the insecticide thiamethoxam on the survival and morphological parameters of the stingless bee Melipona scutellaris were investigated in the present study. Cells from both non-target organs (Malpighian tubules and midgut) and target organs (brain) were analyzed for morphological alterations using light microscopy and transmission electron microscopy. The findings showed that when M. scutellaris foragers were exposed to a sublethal concentration of thiamethoxam (LC50/100 = 0.000543 ng a. i./µL), longevity was not reduced but brain function was affected, even with the non-target organs attempting to detoxify. The cellular damage in all the organs was mostly reflected in irregular nuclei shape and condensed chromatin, indicating cell death. The most frequent impairments in the Malpighian tubules were loss of microvilli, disorganization of the basal labyrinth, and cytoplasmic loss. These characteristics are related to an attempt by the cells to increase the excretion process, probably because of the high number of toxic molecules that reach the Malpighian tubules and need to be secreted. In general, damages that compromise the absorption of nutrients, excretion, memory, and learning processes, which are essential for the survival of M. scutellaris, were found. The present results also fill in gaps on how these bees respond to thiamethoxam exposure and will be useful in future risk assessments for the conservation of bee biodiversity.

Enzymatic responses in the head and midgut of Africanized Apis mellifera contaminated with a sublethal concentration of thiamethoxam.

The increasing use of insecticides, promoted by the intensification of agriculture, has raised concerns about their influence on the decline of bee colonies, which play a fundamental role in pollination. Thus, it is fundamental to elucidate the effects of insecticides on bees. This study investigated the damage caused by a sublethal concentration of thiamethoxam - TMX (0.0227 ng/µL of feed) in the head and midgut of Africanized Apis mellifera, by analyzing the enzymatic biomarkers, oxidative stress, and occurrence of lipid peroxidation. The data showed that the insecticide increased acetylcholinesterase activity (AChE) and glutathione-S-transferase (GST), whereas carboxylesterase (CaE3) activity decreased in the heads. Our results indicate that the antioxidant enzymes were less active in the head because only glutathione peroxidase (GPX) showed alterations. In the midgut, there were no alkaline phosphatase (ALP) or superoxide dismutase (SOD) responses and a decrease in the activity of CaE was observed. Otherwise, there was an increase in GPX, and the TBARS (thiobarbituric acid reactive substances) assay also showed differences in the midgut. The TBARS (thiobarbituric acid reactive substances) assay also showed differences in the midgut. The results showed enzymes such as CaE3, GST, AChE, ALP, SOD, and GPX, as well as the TBARS assay, are useful biomarkers on bees. They may be used in combination as a promising tool for characterizing bee exposure to insecticides.

The functional activity of the miR-1914-5p in lipid metabolism of the hepatocarcinoma cell line HepG2: a potential molecular tool for controlling hepatic cellular migration.

Hepatocellular carcinoma is one of the most common types of cancer in the world with high mortality rate and new therapies that control of fatty acid metabolism may limit the proliferation of cancer cells. In the last two decades, the non-coding RNAs have been considered as promising molecular tools to treat diseases, because they are able to modulate gene expression and the metabolic routes; however, deep investigation of their mechanistic behavior in pathologies must be performed. Thus, our aim was to evaluate the modulatory effect of the miR-1914-5p in controlling lipid metabolism in HepG2, a widely used human hepatocarcinoma cell line. The molecular and cellular analyses demonstrated that the functional inhibition of the investigated microRNA completely changed the cellular metabolism and behavior, compared to control groups. The in vitro inhibition of the miR-1914-5p increased the energy expenditure pointed in different analyses, decreasing cell doubling time and migration rate verified in wound healing and in the classical transwell chambers invasion assays, which makes the miR-1914-5p a candidate for further translational and preclinical studies to validate its function in controlling metastasis in liver cancer or even treat those diseases.