Monotheca buxifolia plant extract against Rhipicephalus microplus ticks and Sarcoptes scabiei mites. Additionally, we employed a computational approach to identify phytochemicals from the extract that could serve as drug candidates against these ectoparasites. The contact bioassay results demonstrated that the M. buxifolia plant extract exhibited significant efficacy against R. microplus and S. scabiei, with higher concentrations outperforming the positive control acaricide permethrin in terms of mite mortality. Time exposure to the extract also showed a positive correlation with better lethal concentration (LC50 and LC90) values. Similarly, the adult immersion test revealed a notable inhibition of tick oviposition via the plant extract, especially at higher concentrations. The two-protein primary structure, secondary structure and stability were predicted using the Expasy’s ProtParam server, SOPMA and SUSUI server, respectively. Using Homology modeling, the 3D structure of the protein was obtained and validated through the ERRAT server, and active sites were determined through the CASTp server. The docking analysis revealed that Alpha-Amyrenyl acetate and alpha-Tocopherol exhibited the highest docking scores for S. scabiei and R. microplus aspartic protease proteins, respectively. These phytochemicals demonstrated strong binding interactions, suggesting their potential as acaricidal drug candidates. In conclusion, the M. buxifolia plant extract displayed significant acaricidal activity against R. microplus and S. scabiei. Moreover, the computational approach identified promising phytochemicals that could serve as potential drug candidates for controlling these ectoparasites.Tick and mite infestations pose significant challenges to animal health, agriculture, and public health worldwide. The search for effective and environmentally friendly acaricidal agents has led researchers to explore natural alternatives. In this study, we investigated the acaricidal potential of the  Sarcoptes scabiei, an astigmatic mite that burrows into the epidermis, actively penetrating the stratum corneum [Sarcoptic mange, also known as scabies in human patients, is an extremely contagious skin condition caused by  corneum . The adu corneum . The dis corneum ,4.Sarcoptic mange is an exceptionally contagious skin disease that spreads through direct skin-to-skin contact, fomites or contact with contaminated environments frequented by heavily affected hosts ,6. NymphRhipicephalus microplus tick (Acari: Ixodidae) is a profoundly impactful tick species with devastating effects on bovine well-being and productivity. Its widespread presence in various tropical and subtropical regions worldwide, particularly in Pakistan and India [Babesia bigemina and Anaplasma marginale, which cause economically significant and very deadly illnesses, particularly in high-yielding crossbred and exotic cattle herds [The nd India , poses ale herds . Ticks ale herds . TherefoMonotheca buxifolia, a member of the Sapotaceae family, is a very important indigenous evergreen tree or thorny shrub known for its tasty fruits that grows in a variety of habitats including northwest Pakistan, Afghanistan, and Iran. This excellent plant performs a variety of essential functions, including feed, fuel, tiny wood, and fences. Its importance extends to traditional medicine, where the fruit has been valued for its digestive, hematinic, vermicide, and laxative characteristics, and the aerial portions are used to treat rheumatism and urinary tract infections [fections . The plafections ,22,23,24S. scabiei cDNA library, researchers identified a single aspartic protease, and functional assays revealed its ability to digest human “hemoglobin, serum albumin, fibronectin, and fibrinogen” while not affecting collagen III or laminin. The presence of this aspartic protease (SsAP) opens up possibilities for interfering with its function, which may have implications for mite survival [Aspartic protease is an enzymatic protein responsible for catalyzing the hydrolysis of peptide bonds with the aid of a trapped water molecule . These psurvival .M. buxifolia plant extract to inhibit ticks and mites in vitro, as well as to investigate the inhibitory interactions between the plant’s phytochemical and the aspartic protease proteins of ticks and mites using in silico methods. It is worth noting that, prior to our research, there has been limited exploration of M. buxifolia plant extract for its acaricidal potential against these ticks and mites. Furthermore, the specific phytochemicals derived from this plant have not been previously utilized in docking experiments aimed at understanding their interactions with the target proteins in ticks and mites. This novelty adds significance to our study, as it contributes to the emerging knowledge about the potential use of M. buxifolia in pest control and sheds light on the underlying molecular mechanisms.In this context, the present study aimed to assess the potential of M. buxifolia plant extract against the S. scabiei var. cuniculi at varying concentrations were tested in different time intervals, as shown in 50 and LC90 values as shown in 50 value and its corresponding calculated confidence limits were 0.342 (0.202–0.467) g/mL, and the LC90 value and its corresponding confidence limits were 1.529 (1.037–3.429) g/mL. Similarly, the lethal time required to kill 50% and 90% (LT50 and LT90) of the test mites’ populations at an extract concentration of 2 g/mL was 0.931 (0.673–1.192) and 3.356 (2.474–5.479) h, respectively, as shown in The efficacy of the M. buxifolia plant extract in inhibiting the oviposition (IO) of ticks was assessed at different concentrations. The results revealed a significant inhibition of egg laying by ticks when exposed to the extract. At a 40 mg/mL concentration, the extract displayed a substantial IO of 35.612%, demonstrating its potential to hinder the reproductive capabilities of ticks. Lower concentrations of 20 mg/mL, 10 mg/mL, 5 mg/mL, and 2.5 mg/mL also exhibited notable IO values of 30.574%, 15.960%, 10.239%, and 1.326%, respectively , the efficacy of ectively , Table 450 and LC90) values of M. buxifolia plant extract against R. microplus in vitro using the AIT. At 24 h, the extract exhibited an LC50 value of 48.678 mg/mL, indicating the concentration required to cause 50% mortality, while the LC90 value was 85.498 mg/mL, representing the concentration required for 90% mortality. At 48 h, the extract showed a significantly lower LC50 value of 3.013 mg/mL and an LC90 value of 43.759 mg/mL, indicating its increased potency over time. The statistical analysis confirmed significant differences in effectiveness between the concentrations and time points (p < 0.05).50 and LT90) values for M. buxifolia extract against R. microplus in vitro using the AIT. The LT50 values ranged from 24.77 h (at 40 mg/mL concentration) to 60.179 h (at 2.5 mg/mL concentration), signifying the time required to achieve 50% mortality. Similarly, the LT90 values ranged from 49.446 h (at 40 mg/mL concentration) to 226.023 h (at 2.5 mg/mL concentration), representing the time needed to attain 90% mortality. The statistical analysis did not reveal significant differences between the concentrations (p > 0.05).M. buxifolia plant extract exhibited concentration-dependent effects on the lethal concentrations and lethal time in the AIT against R. microplus. The extract demonstrated significant potency in inducing mortality, with lower concentrations and longer exposure times showing greater effectiveness.These results indicate that the R. microplus and S. scabiei mite was analyzed using the highly reliable Expasy ProtParam server. The S. scabiei aspartic protease protein was found to be 419 amino acids long with a molecular weight of 46,274.01, while the R. microplus protein was 391 amino acids long with a molecular weight of 42,221.45. The S. scabiei protein had 36 positively charged residues (Arg + Lys) and 35 negatively charged residues (Asp + Glu), indicating its significance in potential interactions. On the other hand, the R. microplus protein exhibited 31 positively charged residues and 29 negatively charged residues, which still showcased its potential functional importance. The computed GRAVY (“Grand Average of Hydropathicity”) values further emphasized the distinct nature of these proteins. The S. scabiei protein displayed an incredibly low GRAVY score of 0.003, highlighting its highly hydrophilic nature. In contrast, the R. microplus protein showed a relatively higher GRAVY score of 0.105, signifying its better interaction with water and implying a more hydrophilic characteristic. To ascertain the stability of these proteins, the instability index (II) was computed. Remarkably, both target proteins exhibited stability with II values of 33.04 for S. scabiei and 32.30 for R. microplus, as shown in The primary structure of aspartic protease proteins from S. scabiei aspartic protease protein. On the other hand, the R. microplus aspartic protease protein exhibited 22.51% alpha helices, 40.15% random coils, 31.46% extended strands, and 5.88% beta-turns. R. microplus and S. scabiei.The structure of a protein is intimately linked to its function, and this connection is particularly evident in its secondary structure, which includes helices, sheets, turns, and coils. These elements play a crucial role in defining the protein’s overall structure, function, and interactions . For thehttps://harrier.nagahama-i-bio.ac.jp/sosui/, accessed on 22 May 2023). This framework, which combines amphiphilic amino acids, was utilized for isolating coat proteins and estimating transmembrane helical regions. For soluble proteins and membrane proteins, amino acid sequences were considered based on a sequence identity cutoff of 25%. Natural occurrences revealed the presence of lysine, arginine, tyrosine, and tryptophan amino acids near the ends of transmembrane helices. Amphiphilicity values are positive for polar residues with a long hydrophobic stem beyond the γ carbon, whereas amphiphilicity values are 0 for tiny polar and hydrophobic residues [The SOSUI server effectively identified transmembrane regions for various proteins. By submitting a protein of interest, this server categorizes it as cytoplasmic or transmembrane. To enhance transmembrane helix prediction, an amphiphilic list of amino acid sequences was generated using the SOSUI server tool , validated that both proteins are 100% membrane proteins. The SOSUI signal anticipated that both proteins would include a signal peptide. SOSUI gramN predicted that the cytoplasm is the subcellular localization site of S. scabiei aspartic protease while the subcellular localization site of R. microplus aspartic protease protein is outer membrane (SOSUI server tool results show that both membrane .S. scabiei and R. microplus. Only one model was generated with a GMQE of 0.88 and the best available template (2psg.1.A with 43.29% sequence identity) to build a 3-D model for S. scabiei aspartic protease and 0.68 GMQE  for Rhipicephalus microplus aspartic protease. We utilized SAVESv6.0 to compare each model’s ERRAT value and Ramachandran plot, as shown in S. scabiei aspartic protease in the model has two residues in the forbidden area, but the same protein in R. microplus contains none. Validation with Ramachandran plot in SAVESv6.0 shows that the ERRAT value for the aspartic protease protein in S. scabiei is 82.703, whereas it is 80.719 for the aspartic protease protein in R. microplus  and R. microplus aspartic protease protein  as shown in CASTp was used to find the key residues and region around the binding cavity of The design of structure-based drugs is highly reliant on docking small molecule compounds into a receptor’s binding site and estimating the complex’s binding affinity. AutoDock Vina is a free and open-source molecular-docking, virtual-screening, and drug-discovery tool with multicore capability, lightning-fast processing rates, and an intuitive user interface. When the structure of the ligand–protein complex is known, the docking tool’s ability to imitate the ligand’s binding mode to the protein may be assessed. The root mean square deviation (RMSD) between the docked position and the ligand’s crystallographically observed binding site is widely used as a criterion, and a value less than 2 Å is typically regarded as successful.S. scabiei aspartic protease and the R. microplus aspartic protease protein. The active-site residues of both the protein making different numbers of hydrogen bonds and the hydrophobic bonds were identified. In this study, S. scabiei aspartic protease and R. microplus aspartic protease proteins were thus docked with the selected compounds to assess the binding interactions. Alpha-Amyrenyl acetate interacts with the S. scabiei receptor via a series of amino acid residues—the hydrogen bond (Tyr 329), pi-alkyl (Ala 50 and Tyr 329), and alkyl —with the docking score of −7.3 Kcal/mol. The ranking of the docking score is as follows: Alpha-Amyrenyl acetate > Lupenol > Butelin > 3-Deoxyestradiol > cis-13,14-Epoxydocosanoic acid > Indole > alpha-Tocopherol > 5-Hydroxytryptamine > Ascorbyl 6-stearate > 3,4,4-Trimethyl-5-pyrazolone. Furthermore, the binding interactions of compounds and R. microplus aspartic protease proteins is as follows: Alpha-Tocopherol > Alpha-Amyrenyl acetate > 3-Deoxyestradiol > Lupenol > Butelin > 5-Hydroxytryptamine > Ascorbyl 6-stearate > Indole > cis-13, 14-Epoxydocosanoic acid > 3,4,4-Trimethyl-5-pyrazolone. Alpha-Tocopherol binds to the R. microplus aspartic protease protein via a series of bonds: hydrogen bond (Gly 139), pi-Alkyl and Alkyl , Pi-Pi stacked(Tyr 134), and Pi-Anion (Asp276). The summaries of the docking study are shown in CASTp was used to find the key residues and region around the binding cavity of the S. scabiei aspartic proteinase protein and X: 21.6564 Y: −10.6987 Z: 22.5169 Å for the R. microplus aspartic proteinase protein. We carefully examined the interactions of the docking poses with the active site residues. Ultimately, we selected the pose with the highest binding affinity, which was determined to be −7.1 and −6.8 kcal/mol for S. scabiei aspartic protease and R. microplus aspartic protease, respectively. This validation exercise was deemed successful because the docked complexes precisely replicated the original ligand poses, matching the native ligands with RMSD values of 1.83 Å S. scabiei aspartic proteinase protein , and a potential grid box was created using AutoGrid4 with a spacing of 0.375 Å, approximately one-fourth of the length of a C-C covalent bond. The dimensions of the grid box were centered on X: 21.8344 Y: −5.6644 Z: 19.7712 Å for the  protein  and 1.04 protein .The necessity for tick management puts the dairy industry’s viability at risk in locations where ticks thrive and proliferate . The samS. scabiei mites, whose mortality was also time- and dose-dependent. These results are in line with the findings of [S. scabiei mites and R. microplus compared with the negative control.The current study revealed that the mean mortality of adult ticks was increased significantly with increased dosage (concentration) and exposure time after in vitro treatment for the tested botanicals. The same was the case for dings of ,38, in wS. scabiei mites and R. microplus were found to play an essential role in the survival and sustenance of the parasites by facilitating the breakdown of host hemoglobin during the blood-feeding process [M. buxifolia have been shown to meet all the requirements for classification as pharmaceuticals. Thus, the aforementioned phytochemicals were used in docking experiments with the aspartic protease parasite enzymes. Since we wanted to stop these specific enzymes from working, we bound inhibitors to their active sites. All of the phytochemicals were shown to have stronger affinities to the active pockets and lower binding energies compared to the synthetic drug. Furthermore, the existence of typical hydrogen bonds in the protein–ligand complex’s 2D bond contacts demonstrated robust binding. Aspartic protease inhibitory action was shown by plant-derived compounds with significant affinities, such as Alpha-Amyrenyl acetate against R. microplus and alpha-Tocopherol against S. scabiei.Lead and target identification, followed by lead optimization, was a lengthy and costly process in the traditional drug development cycle. In the modern age, computational biology has created a low-cost, rapid method for finding novel drugs . Potenti process ,40. TherM. buxifolia were collected from Abdul Wali Khan University Mardan’s (AWKUM) botanical garden in Khyber Pakhtunkhwa . The plant materials were inspected for damage and rinsed. The leaves were then taken to the herbarium of the Department of Botany, AWKUM, and assigned the accession number Awkum.Bot. They were then air-dried for 2 weeks. After 15 days, the dried leaves were ground into powder, and stock solutions were prepared as described by [Aerial parts from S. scabiei var cuniculi.Mites were collected from rabbits at AWKUM’s rabbit farms, where hay served as bedding and was changed daily. Upon detecting signs of mange, skin scraps were taken from the rabbits using the method described by  and immeR. microplus ticks was conducted from cattle and buffaloes at various farms located in Mardan, Khyber Pakhtunkhwa, with precise coordinates provided as 34.1986° N, 72.0404° E. Subsequently, the ticks underwent a comprehensive cleaning process and were morphologically identified as R. microplus through the utilization of standard tick identification keys, employing a microscope [In strict adherence to the guidelines outlined by the World Association for the Advancement of Veterinary Parasitology  a carefucroscope . A totalThe study was first approved by the ethical committee of AWKUM. Skin scraps were taken from the infested rabbits raised at the rabbit form adhering to the Guide for the Care and Use of Laboratory Animals (8th edition) . The infR. microplus ticks was determined using the adult immersion test (AIT) and larval packet test (LPT) according to the protocol outlined by Ayub et al, [IO) using the following formula [The extracts efficacy against b et al,  and Matob et al, . The tic formula :% IO=EggS. scabiei (accession numbers: V5NEJ5) and R. microplus aspartic proteinase protein with accession number C3UTE0 were obtained from the UniProtKB database. Both the protein sequences were retrieved in FASTA format and used for further analysis.The amino acid sequences of aspartic protease protein from The protein’s physicochemical characterization required the calculation of many critical characteristics using well-established methodologies. Expasy’s ProtParam server  was usedhttps://harrier.nagahama-i-bio.ac.jp/sosui/, accessed on 22 May 2023 [R. microplus and S. scabiei mite. These indices were used to estimate whether the protein was in the cytoplasm or bridged the transmembrane. Furthermore, the SOSUI server’s numerous tools, such as SOSUI (Batch), SOSUIsignal, SOSUIgramN, and SOSUImp1, were used to predict parts of the secondary structure of the aspartic protease protein from its supplied amino acid sequence [In this study, the SOSUI server (May 2023 ) was empsequence . This prhttps://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html, accessed on 22 May 2023 [The SOPMA tool (May 2023 ) was utihttps://swissmodel.expasy.org/, accessed on 25 May 2023) for prediction of the 3D structure model using automated comparative modeling as described by [https://saves.mbi.ucla.edu/, accessed on 26 May 2023, which is a comprehensive platform for protein structure validation. The Ramachandran plot was constructed using the PROCHECK server [https://services.mbi.ucla.edu/ERRAT, accessed on 1 June 2023). This tool assesses the statistics of nonbonded interactions between different atom types within a 9-residue sliding window [The FASTA sequences of the target proteins were submitted to the SWISS-MODEL server  has proven to be an immensely valuable and indispensable tool for determining the active sites of proteins. Through CASTp, a highly detailed, comprehensive, and quantitative analysis of the topographical features of proteins is achieved. This analysis enables the precise localization and measurement of active pockets on both the protein’s surface and its interior 3D structure. As a result, CASTp facilitates the accurate prediction of critical regions and key residues within the protein that play a significant role in interacting with ligands [The use of the Computed Atlas of Surface Topography of Proteins (CASTp) server . The docking process aimed to explore a range of possible conformations and orientations for the ligands at the binding sites. Prior to docking, the protein structures were prepared in PyRx (version 0.8) software, generating PDBQT files with added hydrogen atoms to all polar residues. The ligands’ bonds were set to be rotatable to enhance flexibility during docking. To carry out the protein-fixed ligand-flexible docking, the Lamarckian Genetic Algorithm (LGA) method was employed for all calculations. The binding site on the target proteins, S. scabiei aspartic proteinase protein and R. microplus aspartic proteinase protein, was defined by establishing a grid box with a grid spacing of 0.375 Å. For the S. scabiei aspartic proteinase protein, the grid box was centered on coordinates X: 21.8344, Y: −5.6644 and Z: 19.7712 Å, while for the R. microplus aspartic proteinase protein, it was centered on coordinates X: 21.6564, Y: −10.6987 and Z: 22.5169 Å. To ensure the reliability of the results, ten runs with AutoDock Vina were performed for each ligand structure in all cases, saving the best pose from each run. The final affinity value was determined by averaging the affinity of the best poses obtained in the ten runs. The analysis of protein–ligand conformations, including interactions such as hydrogen bonds and bond lengths, was conducted using PyMol (version 2.5.4) and Discovery Studio Visualizer (version 4.5), allowing for a comprehensive examination of the complex structures and their interactions.The binding mode and interaction of target proteins with individual chemical constituents of The validation of our docking protocol aimed to establish its precision and reliability in replicating the binding model and molecular interactions observed in experimentally modeled protein structures during our current in silico studies. To validate the procedure, we utilized an aspartic protease inhibitor as a test case. The validation process involved manually removing modeled proteins and redocking the inhibitor into the active site using AutoDock Vina (version 1.1.2) software. This entailed extracting inhibitor heteroatoms from the protein complex, saving them as a separate inhibitor in PDB format, while keeping the grid parameters consistent. This rigorous validation ensured the accurate binding of the inhibitor to the active site cleft, with minimal deviation compared to the actual co-crystallized complex. Subsequently, we superimposed the redocked complex onto the reference co-crystallized complex using PyMOL 2.3, calculated the root mean square deviation (RMSD), and generated a 2-dimensional image highlighting the relevant amino acid residues with Discovery Studio (version 4.5) software. This process ensured the reliability of our docking methodology and its fidelity in reproducing binding characteristics observed in experimental crystallographic data.50, LC90 and LT50, LT90) were calculated in R using the “ecotox” package, and all the data were graphically presented using “ggplot2 and ggpubr” R package.All statistical approaches were made in R and RStudio. The data were first arranged in Microsoft Excel (v 2302) and then imported into the R working environment for further statistical analysis. Descriptive statistics of the data were calculated and presented in a table with mean ± standard deviation. The significant difference between the different concentrations were calculated using one-way ANOVA  followed by the Tukey honesty significance difference (HSD) test. Furthermore, 50% and 90% lethal concentration and lethal time (LCM. buxifolia plant possesses notable acaricidal properties, effectively eliminating ticks and mites. Moreover, our computational approach has identified promising phytochemicals with the potential to serve as viable drug candidates against S. scabiei and R. microplus. These findings warrant further investigation through clinical trials, paving the way for the development of an effective acaricidal drug. The robust efficacy of the most potent chemicals within the plant extract positions them as integral components of a comprehensive strategy to combat R. microplus ticks and S. scabiei mites.Based on the study conducted, we can confidently assert that the


COVID-19 pandemic's disproportionate impact on childhood bereavement for youth of color: reflections and recommendations By Burns M, Landry L, Mills D, Carlson N, Blueford JM, and Talmi A. (2023) Front. Pediatr. 11:1063449. doi: 10.3389/fped.2023.1063449A Corrigendum on Text CorrectionIn the published article, there was an error. A parenthetic reference to one of the results is incorrect.Results, Table 2, Paragraph 1. This sentence previously stated:A correction has been made to “Although non-Hispanic white children saw the largest increase in newly bereaved children in 2020 , the findings are more nuanced.”The corrected sentence appears below:“Although non-Hispanic white children saw the largest increase in newly bereaved children in 2020 , the findings are more nuanced.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Lactiplantibacillus plantarum YRL45 was orally administered to mice. The results revealed that it promoted the release of cytokines and improved the phagocytic activity of peritoneal macrophages to activate the immune regulation system. L. plantarum YRL45 was conducive to maintaining the morphology of colon tissue without inflammation and increasing the ratio of villus height to crypt depth in the ileum. The gene expression levels of Muc2, ZO-1 and JAM-1 were significantly up-regulated in the ileum and colon, and the gene expression of Cramp presented an upward trend with L. plantarum YRL45 intervention. Moreover, L. plantarum YRL45 remarkably enhanced the levels of immunoglobulins sIgA, IgA and IgG in the intestine of mice. The 16S rRNA gene analysis suggested that L. plantarum YRL45 administration up-regulated the relative abundance of the beneficial bacteria Muribaculaceae and Akkermansia, down-regulated the abundance of the pathogenic bacteria Lachnoclostridium, and promoted the production of acetic acid, propionic acid and total short-chain fatty acids (SCFAs) in mice feces. Our findings indicated that L. plantarum YRL45 had the potential to be developed as a novel probiotic to regulate the intestinal barrier by altering gut microbiota to enhance intestinal immunity and ameliorate intestinal flora balance.Bacteriocins production is one of important beneficial characteristics of probiotics, which has antibacterial property against intestinal pathogens and is helpful for regulating intestinal flora. To investigate the impact of bacteriocin-producing probiotics on gut microecology, bacteriocin-producing  Bifidobacteria are important sources of probiotics, which are widely used in the food and pharmaceutical industries to regulate immune function and protect the intestinal barrier, thereby improving enteritis, obesity, diabetes and other diseases . I. IL. pla obesity , so regu > 0.05) I, and thresponse . Lachnocpatients . The rel > 0.05) J, which L. plantarum YRL45 had a significant impact on the functions of intestinal microorganisms in aminoacyl-tRNA charging (p < 0.05), aromatic compound biosynthesis (p < 0.05), carbohydrate biosynthesis (p < 0.05), cofactor, prosthetic group, electron carrier, and vitamin biosynthesis (p < 0.01), nucleic acid processing (p < 0.05), and tRNA charging (p < 0.05), which showed that L. plantarum YRL45 modulated the transcription, translation and biosynthesis of intestinal microflora and stimulated body growth and metabolism.Statistical analysis of the KEGG metabolic pathway was carried out for mice intestinal flora. It can be seen from L. plantarum YRL45 on the intestinal microflora of mice, the changes of SCFAs were further determined. The results showed that oral administration of L. plantarum YRL45 significantly up-regulated the levels of acetic acid (p < 0.05), propionic acid (p < 0.05) and total fecal SCFAs (p < 0.01)  A, which relation B. Zhao em et al.  reportednd SCFAs . Our finL. plantarum YRL45 was conducted. In Muribaculaceae and Akkermansia were correlated positively with the gene expression of Muc2, ZO-1, JAM-1 and Cramp in the ileum and colon of mice, while Parabacteroides and Lachnoclostridium were correlated negatively. Similarly, Schneider et al. [Akkermansia. Previous research demonstrated that a negative correlation between Lachnoclostridium and Muc2 expression was observed [Parabacteroides abundance was correlated negatively with the level of ZO-1, which could affect the intestinal barrier [L. plantarum YRL45 regulated the gene expression of intestinal mucin, tight junction proteins and endogenous antimicrobial peptide by affecting intestinal flora, thereby enhancing intestinal immunity, improving intestinal microecological balance and promoting body health.The association of intestinal microbes and four key intestinal genes in mice gavaged with r et al.  reportedobserved . It was  barrier . TherefoL. plantarum YRL45 on gut microecology in mice was investigated. L. plantarum YRL45 demonstrated immunomodulatory abilities in increasing cytokine levels and peritoneal macrophages phagocytic activity. In addition, the strain was conductive to promoting intestinal mucin expression, improving intestinal tissue morphology, and exerting tight junction intestinal barrier function, which also promoted the production of immunoglobulins and stimulated the expression of endogenous antimicrobial peptide in the intestine. The regulation mechanism of L. plantarum YRL45 on the gut might be associated with promoting favorable changes in intestinal flora and the production of SCFAs. Our findings highlighted that bacteriocin-producing L. plantarum YRL45 enhanced intestinal immunity and modulated gut microbiota, which could be considered a valuable probiotic for ameliorating intestinal microecology and facilitating gut health.In this study, the influence of bacteriocin-producing


Meliponini) are a ubiquitous and diverse element of the pantropical melittofauna, and have significant cultural and economic importance. This review outlines their diversity, and provides identification keys based on external morphology, brief accounts for each of the recognized genera, and an updated checklist of all living and fossil species. In total there are currently 605 described extant species in 45 extant genera, and a further 18 extinct species in nine genera, seven of which are extinct. A new fossil genus, Adactylurina Engel, gen. nov., is also described for a species in Miocene amber from Ethiopia. In addition to the systematic review, the biology of stingless bees is summarized with an emphasis on aspects related to their nesting biology and architecture.Stingless bees ( Meliponini who we had the great pleasure of knowing: Jesus S. Moure (1912–2010), Charles D. Michener (1918–2015), and João M.F. de Camargo (1941–2009). Their monumental efforts toward revealing the fundamentals of stingless bee biology, morphology, phylogeny, and evolution will never be surpassed. All of our work builds on their strong foundation.We dedicate this small contribution to the memory of three titans of the In addition, we dedicate this work to our dear friend and colleague Fernando A. Silveira (1960–2022), whose untimely passing in August 2022 deprived melittology of one of its kindest and most generous scholars. He is greatly missed.Meliponini , bumble bees (Bombini), orchid bees (Euglossini), and, of course, the stingless bees (Meliponini). They are managed for their honey, second only to the honey bees, and are growingly used for agricultural purposes  proximally on the metabasitarsus, the simple pretarsal claws, alar venation reduction, and, of course, the general reduction of sclerites associated with the sting complex. Other features that in combination help to define Meliponini but are found in other combinations among the remaining corbiculate tribes are the general reduction of outer grooves on the mandibles, the loss of the metatibial spurs, the presence of arolia, the absence of a supra>-alar carina, and the presence of a jugal lobe on the hind wing , in either a single genus, Melipona, or into two genera, Melipona Illiger and Trigona Jurine. It was not until the works of Heinrich Friese (1860–1948) and Theodore D.A. Cockerell (1866–1948) that serious alterations to the classification were initiated, although most species were still placed in a massive, ill-circumscribed Trigona. These two authors were then followed by the extensive and detailed studies of Herbert F. Schwarz (1883–1960) and Jesus S. Moure (1912–2010), who effectively provided new interpretations for natural groupings of stingless bees, with the latter establishing the foundations for our current system of Meliponini. The growing biological data available for many species also served as a new source of character information for establishing groupings of these bees . These finer points are all worthy of merit and undoubtedly will be revised in time. For now, we have followed the system as recently outlined in Mourella as a genus and some additions from the last year  and herein (one new genus). Although we allude to some disparities and future subjects to be resolved, we do not intend here to emphasize justification or alternative interpretations, which are more fully explained in some of the recent publications cited herein. Regardless, the revised classification is based on comprehensive phylogenetic hypotheses for the tribe  and Argentina (Buenos Aires) up to 27.03°N in Mexico , at exceptional elevations up to 4000 m.a.s.l. in Peru and Bolivia . Meliponliponina , a groupliponina . As of tPlebeia Schwarz and occurring from southern Mexico to Costa Rica. The species look like smaller species of Plebeia , at only ~ 3 mm length, but can be distinguished by the generally more matte integument and coarser sculpturing. Nesting biology has only been studied for Asperplebeiatica (Wille), which nests in tree cavities and, unlike the superficially similar Plebeia, builds brood clusters rather than combs, although species of Nanoplebeia also build brood clusters A.tica (Wille)Cephalotrigona Schwarz includes modestly large bees (8–10 mm), which are generally dark brown to black with faint yellow marks. Noteworthy for the genus is the carinate to lamellate preoccipital ridge, the abundant facial punctation, the long legs, and spatula- or racket-shaped metatibiae with broad corbiculae and only simple setae on the retromarginal edge. Nests are built in tree hollows, and the entrances are a bee-sized hole on a short, rounded platform, built only with cerumen and dark solid materials. The honey is of good quality, and they usually store propolis in abundance. Colonies are large but they are not easy to manage in meliponiculture. This genus occurs from Colima and Jalisco, as well as in Tamaulipas, Mexico to Santa Catarina, Brazil and Missiones, Argentina. The most common species between Mexico and Colombia is Cephalotrigonazexmeniae (Cockerell), similar in appearance to Trigona (Koilotrigona) fulviventris Guérin-Méneville since both are black with a reddish orange metasoma but is larger, while the most common in South America is C.capitata (Smith). Currently, there are only keys for the species from Mexico and Central America ( America  and a reC.capitata (Smith)C.eburneiventer (Schwarz)C.femorata (Smith)C.oaxacana AyalaC.zexmeniae (Cockerell)Duckeola Moure are robust bees of 8–9 mm length, found in Brazil, Ecuador, French Guiana, and Colombia. Perhaps the most striking feature of Duckeola is the considerably depressed parocular area between the compound eyes and the vertex, which elevates the vertex noticeably. In addition, the metatibia is noticeably claviform and lacks plumose setae on the retromarginal surface, and the mesotibial spur is absent (The two species of s absent . No key D.ghilianii (Spinola)D.pavani (Moure)Plebeia, but have a reticulated matte integument, conspicuous abundant whitish pubescence, particularly on the face, and almost no yellow maculation on the face. The bees occur in southern Brazil and build irregular combs without an involucrum.This is a genus of tiny bees, 3 mm in length, which look much like F.schrottkyi (Friese)Frieseomelitta Ihering includes slender species of 4–7 mm in length, ranging from Sinaloa and Veracruz, Mexico to Mato Grosso do Sul, Brazil. Recognition of the genus is aided by the presence of yellow marks on the face bordering the compound eyes on the paraocular area and genae, the absence of the mesotibial spur, the enlarged and inflated metatibia with a small corbicular depression restricted to the apical third, and an overall spatulate or racket-shaped to claviform metatibia with plumose setae on the retromarginal edge. The shape of the metasoma is subtriangular when constricted, in dorsal view, in the varia species group, while the metasoma is elongate, even when contracted, owing to broader terga II and III (this is the situation in the nigra and portoi species groups). The nigra species group has a claviform metatibia, while the portoi species group has a baseball-bat-shaped metatibia. Adults soon turn black on the head and mesosoma after emergence but the metasoma remains whitish for more than a week. The milky white wing tip in most species is another distinctive feature. The nests are distinctive for the arrangement of brood in clusters. New species are being described and a key to species developed by FFO (pers. obs.).The genus F.dispar (Moure)F.doederleini (Friese)F.flavicornis (Fabricius)F.languida MoureF.lehmanni (Friese)F.longipes (Smith)F.meadewaldoi (Cockerell)F.nigra (Cresson)F.paranigra (Schwarz)F.paupera (Provancher)F.portoi (Friese)F.silvestrii (Friese)F.trichocerata MoureF.varia (Lepeletier)Geotrigona nest in cavities in the ground. The species are generally robust, 5–7 mm in length, with a short broad metasoma. The genus is distributed from Michoacan, in the central Balsas River depression in Mexico to Santiago de Estero, Argentina. As the name of the genus suggests, species of G.acapulconis (Strand)G.chiriquiensis (Schwarz)G.fulvohirta (Friese)G.fumipennis Camargo & MoureG.joearroyoi Gonzalez & EngelG.kaba Gonzalez & SepúlvedaG.leucogastra (Cockerell)G.lutzi Camargo & MoureG.terricola Camargo & MoureG.aequinoctialis (Ducke)G.argentina Camargo & MoureG.fulvatra Camargo & MoureG.kraussi (Schwarz)G.kwyrakai Camargo & MoureG.mattogrossensis (Ducke)G.mombuca (Smith)G.subfulva Camargo & MoureG.subgrisea (Cockerell)G.subnigra (Schwarz)G.subterranea (Friese)G.tellurica Camargo & MoureG.xanthopoda Camargo & MoureLestrimelitta Friese are cleptobiotic , which confuses the defense communication between workers and guards of the host L.galvisi Guevara, Gonzalez, & OspinaL.glaberrima Oliveira & MarchiL.glabrata Camargo & MoureL.guyanensis RoubikL.huilensis Gonzalez & GriswoldL.monodonta Camargo & MoureL.mourei Oliveira & MarchiL.niitkib AyalaL.rufipes (Friese)L.similis Marchi & MeloL.chacoana Roig-AlsinaL.ciliata Marchi & MeloL.limao (Smith)L.maracaia Marchi & MeloL.opita Gonzalez & GriswoldL.piedemontana Gonzalez & RasmussenL.rufa (Friese)L.spinosa Marchi & MeloL.sulina Marchi & MeloL.tropica Marchi & MeloMelipona includes almost all of the most massive meliponines. These robust bees, 9–14 mm in length, with abundant plumose pubescence on the mesoscutum have some superficial resemblance to the largest African Meliponula. The wings are usually short and only reach the posterior end of the metasoma or slightly exceed it. The integument is generally black, but in some species brown or pale brown, with yellow, ivory, or brown areas, while the mesosoma has pale, brown, or dark pubescence, particularly the mesoscutum. The genus can be found from Sinaloa and south of Tamaulipas in Mexico to northern Argentina. Many species are used in meliponiculture, and these were the first ‘semi-domesticated’ bees, subject to multiplication and husbandry in the prehispanic Mayan culture. Nest products such as cerumen and honey have been extensively used through the ages. Melipona is now the largest genus in the tribe, but modern keys are lacking. A key to the species was provided by M.amazonica SchulzM.asilvai MoureM.bicolor LepeletierM.carioca MoureM.carrikeri CockerellM.marginata LepeletierM.obscurior MoureM.ogilviei SchwarzM.picadensis StrandM.puncticollis FrieseM.schencki GribodoM.schwarzi MoureM.torrida FrieseM.tumupasae SchwarzM.ambigua Roubik & CamargoM.beecheii BennettM.compressipes (Fabricius)M.fasciculata SmithM.grandis Guérin-MénevilleM.insularis Roubik & CamargoM.interrupta LatreilleM.quinquefasciata LepeletierM.salti SchwarzM.triplaridis CockerellM.baeri VachalM.favosa (Fabricius)M.lunulata FrieseM.lupitae AyalaM.mandacaia SmithM.orbignyi (Guérin-Méneville)M.peruviana FrieseM.phenax CockerellM.quadrifasciata LepeletierM.subnitida DuckeM.variegatipes GribodoM.yucatanica Camargo, Moure, & RoubikM.bradleyi SchwarzM.illustris SchwarzM.micheneri SchwarzM.belizeae SchwarzM.boliviana SchwarzM.brachychaeta MoureM.capixaba Moure & CamargoM.captiosa MoureM.colimana AyalaM.costaricensis CockerellM.cramptoni CockerellM.crinita Moure & KerrM.dubia Moure & KerrM.eburnea FrieseM.fasciata LatreilleM.flavolineata FrieseM.fulva LepeletierM.fuscopilosa Moure & KerrM.illota CockerellM.indecisa CockerellM.lateralis ErichsonM.melanoventer SchwarzM.mimetica CockerellM.mondury SmithM.nebulosa CamargoM.nigrescens FrieseM.panamica CockerellM.paraensis DuckeM.rufescens FrieseM.rufiventris LepeletierM.scutellaris LatreilleM.seminigra FrieseM.solani CockerellM.trinitatis CockerellM.fallax Camargo & PedroM.fuliginosa LepeletierM.titania GribodoPartamona Schwarz and Scaptotrigona. The shape of the metatibia is similar to that of Scaptotrigona, with a broader corbicula and with the characteristic large bristles arising from its surface as in Partamona.This genus of little-known black bees from higher elevations (1400–2700 m) in Costa Rica and western Panama has an appearance superficially resembling M.bivea Roubik, Lobo Segura, & CamargoSchwarziana Moure, distributed in the southern portion of South America: Uruguay, Argentina, Paraguay, and Brazil . The sole species builds its nest in the soil and has architectural features typical to all other obligatory ground-nesting stingless bees Scaptotrigona, there is a polished mediolongitudinal depression on the anterior margin and a prominent, deep, U- or V-shaped notch in the apical margin medially. The genus extends from Sonora , Mexico — the farthest north in Mexico for any stingless bee lineage — to the south of Brazil (Rio Grande do Sul). The bees nest in holes of all kinds, including those in the ground as well as in trees. Despite storing comparatively little honey, some species show potential for agricultural pollination and are easy to manage in meliponiculture for this purpose. The genus was revised by This genus includes small bees (4–4.5 mm) with largely black integument and areas of yellow maculation on the mesoscutum, mesoscutellum, and legs. The punctation of the head and mesosoma is quite coarse. The mesoscutellum is projected posteriorly over the metanotum and, like the genus N.dutrae (Friese)N.schultzei (Friese)N.camargoi Rasmussen & GonzalezN.chapadana (Schwarz)N.gaboi Jaramillo, Ospina, & GonzalezN.melanocera (Schwarz)N.mellaria (Smith)N.minuta (Lepeletier)N.occidentalis Jaramillo, Ospina, & GonzalezN.perilampoides (Cresson)N.pilosa Jaramillo, Ospina, & GonzalezN.punctata (Smith)N.testaceicornis (Lepeletier)N.tristella CockerellNogueirapis Moure differs from the closely related Partamona in the presence of abundant yellow markings; only slightly spoon-shaped, not greatly enlarged metatibia of the worker; as well as the smaller body size (3.5–5.5 mm), superficially resembling species of Plebeia. Like Partamona, there are one or two elongate bristles arising from the corbicular surface. Species nest in ground cavities but some also occasionally nest in tree hollows. A key to the species was presented by N.batistai NogueiraN.butteli (Friese)N.costaricana Ayala & EngelN.minor (Moure & Camargo)N.mirandula (Cockerell)N.rosariae NogueiraThis genus includes the infamous “fire bees”, so named because of their characteristic defense system. Workers have well-developed mandibular glands that produce a secretion containing formic acid, and which can inflict significant burns to the recipient of an attack. The bees are orange to black in color, with the integument of the head quite smooth and polished, sometimes with a vitreous appearance. Overall, the bees are typically 5–6 mm in length, with the head large, wider in comparison to the mesosoma, with an enlarged malar space and an interocular distance greater than the length of the compound eye itself. Species range from Chiapas, Mexico to southern Brazil (Santa Catarina). A key to the species was provided by O.chocoana Gonzalez & RoubikO.daemoniaca CamargoO.flaveola (Friese)O.huaoranii Gonzalez & RoubikO.ignis CamargoO.isthmina Gonzalez & RoubikO.mediorufa (Cockerell)O.mellicolor (Packard)O.mulfordi (Schwarz)O.obscura (Friese)O.tataira (Smith)Paratrigona Schwarz are small, between 3.5–5.5 mm in length, and are generally black with well-delimited yellow markings. The mesoscutellum projects posteriorly over the metanotum as a plate, and the metasoma is typically robust, almost as wide as the mesosoma. The genus occurs from Veracruz, Mexico to northern Argentina , and southern Brazil (Rio Grande do Sul). Some species nest on the ground, while others build nests in various substrates, including wood and termite mounds and often on vines and within epiphytic plants. A key to the species was provided by Species of P.impunctata (Ducke)P.isopterophila (Schwarz)P.anduzei (Schwarz)P.catabolonota Camargo & MoureP.compsa Camargo & MoureP.crassicornis Camargo & MoureP.eutaeniata Camargo & MoureP.euxanthospila Camargo & MoureP.femoralis Camargo & MoureP.glabella Camargo & MoureP.guatemalensis (Schwarz)P.guigliae MoureP.haeckeli (Friese)P.incerta Camargo & MoureP.intermedia Oliveira, Madella-Auricchio, & FreitasP.lineata (Lepeletier)P.lineatifrons (Schwarz)P.lophocoryphe MoureP.lundelli (Schwarz)P.melanaspis Camargo & MoureP.myrmecophila MoureP.nuda (Schwarz)P.onorei Camargo & MoureP.opaca (Cockerell)P.ornaticeps (Schwarz)P.pacifica (Schwarz)P.pannosa MoureP.peltata (Spinola)P.permixta Camargo & MoureP.prosopiformis (Gribodo)P.rinconi Camargo & MoureP.scapisetosa Gonzalez & GriswoldP.subnuda MoureP.uwa Gonzalez & VélezP.wasbaueri Gonzalez & GriswoldParatrigona or Plebeia with a dull integument, but has the keirotrichiate area of the metatibia not depressed on the superior margin; has yellow markings on the paraocular area, frontal median line, and spots below the lateral ocelli; and the upper part of the preoccipital ridge lamellate and bordered by a row of robust setae. The genus includes a single species of ~ 4.7 mm in length and is currently known only from Colombia. The nesting biology remains to be documented.This genus superficially resembles P.mayri Camargo & RoubikPartamona are usually 5–6.5 mm in length, with a smooth and shiny integument, which can be black to orange-yellow, depending on the species, with vitreous yellow paraocular markings. The metatibia is quite large and broadened, making it distinctively spoon-shaped, and lacks plumose setae on the retromarginal edge. Species build semi-exposed nests on either natural or human constructions or on trees, as well as in the ground. Nest entrances are built of a material similar to hardened mud, often with a wide entrance. Partamona are not appropriate for meliponiculture as they are quite defensive and challenging to work with, in addition to insignificant amounts of stored honey. The genus occurs from Sonora, Mexico to southern Brazil (Rio Grande Do Sul). The species with testaceous bodies were revised by Species of P.brevipilosa (Schwarz)P.zonata (Smith)P.aequatoriana CamargoP.ailyae CamargoP.auripennis Pedro & CamargoP.batesi Pedro & CamargoP.bilineata (Say)P.chapadicola Pedro & CamargoP.combinata Pedro & CamargoP.criptica Pedro & CamargoP.cupira (Smith)P.epiphytophila Pedro & CamargoP.ferreirai Pedro & CamargoP.grandipennis (Schwarz)P.gregaria Pedro & CamargoP.helleri (Friese)P.littoralis Pedro & CamargoP.mourei CamargoP.mulata MoureP.musarum (Cockerell)P.nhambiquara Pedro & CamargoP.nigrior (Cockerell)P.orizabaensis (Strand)P.pearsoni (Schwarz)P.peckolti (Friese)P.rustica Pedro & CamargoP.seridoensis Pedro & CamargoP.sooretamae Pedro & CamargoP.subtilis Pedro & CamargoP.testacea (Klug)P.vicina CamargoP.vitae Pedro & CamargoP.xanthogastra Pedro & CamargoP.yungarum Pedro & CamargoPlebeia is a diverse group of often small and medium size bees (2–7 mm), with shiny integument bees and prominent yellow or white maculation. The metatibia is triangular shaped with only simple setae on the retromarginal edge, and the keirotrichiate zone of the retrolateral surface extends to the superior margin (without a shiny depressed rim). Included herein is the former genus Plectoplebeia Melo, an apparent synonym of the large subgenus Plebeia s.str. and representing merely larger, higher-elevation, seemingly cloud-forest specialized species of the subgenus , P.aurantia (Engel), P.plectoforma Engel, P.hyperplastica Engel, and P.tigris Engel] (Cecropia Loefl. (Urticaceae), made available by ants, and interact with obligate inquiline scale insects that provide wax and honeydew P.margaritae MoureP.minima (Gribodo)P.orphne EngelP.pleres EngelP.alvarengai MoureP.amydra EngelP.aurantia (Engel), comb. n.P.catamarcensis (Holmberg)P.cora AyalaP.deceptrix EngelP.droryana (Friese)P.emerina (Friese)P.emerinoides (Silvestri)P.flavocincta (Cockerell)P.frontalis (Friese)P.fulvopilosa AyalaP.goeldiana (Friese)P.grapiuna Melo & CostaP.guazurary Alvarez, Rasmussen, & AbrahamovichP.hyperplastica EngelP.jatiformis (Cockerell)P.julianii MoureP.kerri MoureP.llorentei AyalaP.lucii MoureP.malaris MoureP.manantlensis AyalaP.mansita Alvarez & RasmussenP.melanica AyalaP.meridionalis (Ducke)P.mexica AyalaP.molesta (Puls)P.mosquito (Smith)P.mutisi EngelP.nigriceps (Friese)P.nigrifacies (Friese)P.parkeri AyalaP.peruvicola MoureP.phrynostoma MoureP.plectoforma EngelP.poecilochroa Moure & CamargoP.pulchra AyalaP.remota (Holmberg)P.roubiki EngelP.saiqui (Friese)P.silveirai EngelP.tigris EngelP.tobagoensis MeloP.variicolor (Ducke)P.vidali EngelP.wittmanni Moure & CamargoTetragona Lepeletier & Audinet-Serville in the presence of yellow maculation on the face and the velvety setation of the gena, the presence of a mesotibial spur, and the plumose setae on the retromarginal edge of the metatibia but can be distinguished by the setose basal area to the propodeum, the calviform metatibia with the proximal third more plump, and the larger mandibular teeth. The bees are 7–9 mm in length and extend from Costa Rica, with a noteworthy gap through Panama, thence to Colombia, central Brazil, and Peru. A key for the species was presented by This genus greatly resembles P.camargoi (Moure)P.nordestina (Camargo)P.pilicornis (Ducke)P.lurida (Smith)P.occidentalis (Schulz)P.pereneae (Schwarz)Scaptotrigona is a distinctive group of small to medium-sized bees, 4.5–9 mm, which range in color from orange to black and lack yellow markings except for those on the postgena or elsewhere in one species of the subgenus Sakagamilla Moure [Scaptotrigonaaffabra (Moure)]. The preoccipital ridge is carinate to minutely lamellate and with three distinctive pits dorsally, and often with an interruption laterally. The metatibiae are also quite characteristic, subtriangular in shape and lacking plumose setae on the retromarginal edge, but bordered by an abundance of rather thick, curved bristles. Like Nannotrigona Cockerell, the mesoscutellum has a characteristic longitudinal depression or groove extending from anterior margin medially, but the integument is not as coarsely punctured. While the genus is easy to distinguish, the species are quite complex with considerable variation and the identification of the species can be difficult. The genus was recently organized into a series of subgenera, including a key to these subgeneric lineages  and always build regular horizontal combs. A notable exception is S.subobscuripennis (Schwarz), of Costa Rica and western Panama, which nests in cavities in the ground, and apparently never uses tree cavities. The honey has good flavor and is appreciated by many, and propolis from Scaptotrigona is widely used for medicinal purposes. All species have pungent defensive cephalic secretions characterized as 2-nonal and are quite defensive.lineages . A key tublished  and otheS.ascheri EngelS.mexicana (Guérin-Méneville)S.wheeleri (Cockerell)S.bipunctata (Lepeletier)S.emersoni (Schwarz)S.fimbriata EngelS.subobscuripennis (Schwarz)S.tricolorata CamargoS.fulvicutis MoureS.luteipennis (Friese)S.polysticta MoureS.totobi EngelS.aurantipes EngelS.depilis (Moure)S.guimaraesensis Laroca & AlmeidaS.hellwegeri (Friese)S.jujuyensis (Schrottky)S.nuda EngelS.psile EngelS.stipula EngelS.affabra (Moure)S.marialiceae Laroca & AlmeidaS.pasiphaea EngelS.silviae Engel*S.tubiba (Smith)*S.anaulax EngelS.baldwini EngelS.barrocoloradensis (Schwarz)S.caduceus EngelS.ederi EngelS.extranea EngelS.faviziae EngelS.gonzalezi EngelS.grueteri EngelS.illescasi EngelS.kuperi EngelS.limae (Brèthes), sp. inq.S.macarenensis EngelS.magdalenae EngelS.nigrohirta Nogueira & Santos-SilvaS.ochrotricha (Buysson)S.pectoralis  [includingpanamensis (Cockerell)]S.postica (Latreille)S.rosellae EngelS.santiago EngelS.semiflava EngelS.tatacoensis EngelS.turusiri (Janvier), sp. inq.S.vitorum EngelS.xanthotricha MoureS.yungasensis EngelPlebeia. The metabasitarsi are large and dilated, wider than the corresponding metatibiae and are used for rubbing floral structures to mop up loosened and scattered pollen. Nests are in tree cavities or even within the arboreal nests of nasutitermitine termites, and the bees build brood combs, except for S.latitarsis (Friese) and the species of Schwarzula Moure who build brood clusters and S.longula (Lepeletier) that builds simple vertical, double-sided appressed cell combs S.longula (Lepeletier)S.amazonica Nogueira, Oliveira, & OliveiraS.argyrea (Cockerell)S.aspera Nogueira & OliveiraS.atlantica MeloS.cearensis Nogueira, Santos Júnior, & OliveiraS.coccidophila (Camargo & Pedro)S.timida (Silvestri)Plebeia. A key to species for the subgenus Schwarziana was provided by Chapadapis Engel. Where known, the bees build nests in the ground, with the brood combs arranged in spirals. The genus is found in central-southern Brazil, and in Paraguay and northern Argentina.This genus includes medium-sized bees, 6–8 mm in length, which were at one time placed among S.chapadensis MeloS.bocainensis MeloS.mourei MeloS.quadripunctata (Lepeletier)Tetragona superficially resemble species of Frieseomelitta, as both include relatively long-legged bees, differing mainly by the presence of the mesotibial spur, the velvety pubescence of the gena, the yellow maculation of the head in most species  lacks yellow maculation entirely and species of the handlirschii group only have reddish yellow-brown areas on the clypeus), and the shape of the metatibiae, which are narrower. Species of Tetragona are ~ 5–8 mm in length and occur from Tabasco, Mexico to Uruguay. Species are used in meliponiculture and for supplies of sticky resin materials stored in nests. A key is provided by clavipes species group.The bees of the genus T.atahualpa Nogueira & RasmussenT.beebei (Schwarz)T.clavipes (Fabricius)T.dorsalis (Smith)T.essequiboensis (Schwarz)T.goettei (Friese)T.handlirschii (Friese)T.kaieteurensis (Schwarz)T.korotaii NogueiraT.mayarum (Cockerell)T.mourei NogueiraT.perangulata (Cockerell)T.quadrangula (Lepeletier)T.truncata MoureT.ziegleri (Friese)Tetragonisca Moure are small (4–5 mm), slender bees, with yellow maculation on the lower portion of the face in most species, plumose setae on the retromarginal edge of the metatibia, and have a mesotibial spur (resembling Tetragona). However, the species of Tetragonisca have a basal sericeous area  on the retrolateral surface of the metabasitarsus of workers and lack velvety pilosity on the gena. The metatibia is also rather inflated, with the corbicula reduced to the apical portion of the podite. The genus extends from Veracruz, Mexico to northern Argentina. It should be noted that the widespread and common Tetragoniscaangustula (Latreille) appears to be a complex of species. The systematics of this species should be explored in depth. Nests are built in tree cavities or the ground. No key to species is currently available.Species of T.angustula (Latreille)T.buchwaldi (Friese)T.fiebrigi (Schwarz)T.weyrauchi (Schwarz)Frieseomelitta but have conspicuous setae on the compound eyes (hence the generic name). Species of Trichotrigona Camargo & Moure are likely robber bees but seemingly undertaking isolated, rather than mass, raids. This is a genus of enigmatic stingless bees from northern Brazil. The genus includes bees of 5–6 mm in length, and that superficially resemble T.camargoiana Pedro & CordeiroT.extranea Camargo & MoureTetragona), and can be reddish orange to black in coloration, but always lack yellow maculation. The mandibles have well-developed, prominent teeth (either four or five). The metatibiae are claviform and in one subgenus (Necrotrigona Engel) the corbicula is not developed. Aside from the raised median keirotrichiate area on the retrolateral surface of the metatibia, there is also a basal sericeous area  on retrolateral surface of the metabasitarsus. The biology of Trigona is remarkably varied, perhaps more so than any other genus of Meliponini, and includes obligate necrophages that scavange from carcasses (Necrotrigona). Species nest in squirrel or bird nests as well as in tree cavities, while some nest on the ground among tree roots and others build exposed nests around tree branches. The largest nests are composed of the bees’ own feces, from defecated pollen exines. This group now includes 30 described species but will likely be found to have more than twice as many, like Trigonisca T.williana FrieseT.cilipes (Fabricius)T.lacteipennis FrieseT.mazucatoi AlmeidaT.pellucida CockerellT.dimidiata SmithT.sesquipedalis AlmeidaT.venezuelana SchwarzT.braueri FrieseT.fulviventris Guérin-MénevilleT.guianae CockerellT.albipennis AlmeidaT.fuscipennis FrieseT.crassipes (Fabricius)T.hypogea SilvestriT.necrophaga Camargo & RoubikT.daianeae RibeiroT.juvenili RibeiroT.mandaloriana RibeiroT.permodica AlmeidaT.recursa SmithT.amalthea (Olivier)T.amazonensis (Ducke)T.branneri CockerellT.corvina CockerellT.dallatorreana FrieseT.hyalinata (Lepeletier)T.nigerrima CressonT.pampana StrandT.silvestriana T.spinipes (Fabricius)T.truculenta AlmeidaTrigonisca includes exclusively minute stingless bee species and is the earliest-diverging lineage of extant Neotropical Meliponini. The genus is one of the more widespread groups of New World stingless bees, as well as a relatively commonly encountered group of meliponines. The nesting biology was explored for Trigoniscamepecheu Engel & Gonzalez by Celetrigona Moure is presented by Leurotrigona and Exochotrigona Engel apparently form a grade at the base of Trigonisca s.l. and are classified by some authors as a separate genus. They can be easily distinguished from the other Neotropical minute bees by the smooth, polished, and shiny integument, the absence of a preoccipital carina, and the absence of yellow markings. Many new species have been encountered and are currently being described for the western Amazon (Leurotrigona). A revision of Trigonisca s.str. is needed, but a beginning was made by Dolichotrigona, although this group almost assuredly renders Trigonisca s.str. paraphyletic and is therefore not recognized herein. Nonetheless, for readers who prefer to recognize Dolichotrigona we provide the following couplet that distinguishes those species from Trigonisca s.strictiss.The genus n Amazon . A key tT.euclydiana (Camargo & Pedro)T.hirsuticornis (Camargo & Pedro)T.longicornis (Friese)T.manauara (Camargo & Pedro)T.crispula (Pedro & Camargo)T.pusilla (Moure & Camargo)T.gracilis (Pedro & Camargo)T.muelleri (Friese)T.atomaria (Cockerell)T.azteca AyalaT.bidentata Albuquerque & CamargoT.browni (Camargo & Pedro) [Dolichotrigona]T.buyssoni (Friese)T.chachapoya (Camargo & Pedro) [Dolichotrigona]T.clavicornis (Camargo & Pedro) [Dolichotrigona]T.coephloei (Schwarz)T.discolor (Wille)T.dobzhanskyi (Moure)T.duckei (Friese)T.extrema Albuquerque & CamargoT.flavicans (Moure)T.fraissei (Friese)T.graeffei (Friese)T.hirticornis Albuquerque & CamargoT.intermedia MoureT.longitarsis (Ducke) [Dolichotrigona]T.martinezi (Brèthes) [Dolichotrigona]T.maya AyalaT.mendersoni (Camargo & Pedro) [Dolichotrigona]T.mepecheu Engel & GonzalezT.meridionalis Albuquerque & CamargoT.mixteca AyalaT.moratoi (Camargo & Pedro) [Dolichotrigona]T.nataliae (Moure)T.pediculana (Fabricius)T.pipioli AyalaT.rondoni (Camargo & Pedro) [Dolichotrigona]T.roubiki Albuquerque & CamargoT.sachamiski Alvarez & LuciaT.schulthessi (Friese) [Dolichotrigona]T.tavaresi (Camargo & Pedro) [Dolichotrigona]T.townsendi (Cockerell)T.unidentata Albuquerque & CamargoT.variegatifrons Albuquerque & CamargoT.vitrifrons Albuquerque & CamargoMeliponini are unique and found in a line from Senegal to Eritrea along the southern Sahel southward to KwaZulu-Natal, South Africa. The tribe is also found throughout Madagascar. Currently we recognize 33 extant species in the Afrotropical fauna and eight extant genera A.simpsoni MoureA.erythra (Schletterer)A.ferruginea (Lepeletier)A.kitingae Engel & Solórzano-Kraemer†A.togoensis (Stadelmann)Dactylurina Cockerell is perhaps the most distinctive genus of African stingless bees. As the name implies, the metasoma is elongate, thin, and subclavate, giving it a finger-like shape. The genus occurs from Guinea eastward to the Congo and Uganda and thence southward to Angola. The genus is distinctive for building double vertical combs D.staudingeri (Gribodo)Liotrigona Moure, Hypotrigona Cockerell includes minute stingless bees commonly encountered in Africa from Senegal eastward to Eritrea, and southward to northern South Africa. The genus is distinctive for the broadly rounded distal superior angle of the metatibia and the dull, matte, reticulate to micro-alveolate integument. The genus is also commonly encountered in East African copal and modern resins (Like n resins . Nest man resins .H.araujoi (Michener)H.gribodoi (Magretti)H.kleineri Engel & Solórzano-Kraemer†H.ruspolii (Magretti)H.squamuligera (Benoist)Hypotrigona but differ in the smooth and shiny integument and the presence of a distinct superior distal angle on the metatibia. The monotypic subgenus Cleptotrigona Moure includes a species that is a robber bee on Hypotrigona and other Liotrigona. A single extinct species is also known from Early Miocene amber from Ethiopia L.baleensis Pauly & HoraL.betsimisaraka PaulyL.bitika Brooks & MichenerL.bottegoi (Magretti)L.bouyssoui L.chromensis PaulyL.gabonensis Pauly & Fabre AnguiletL.kinzelbachi KochL.madecassa (Saussure)L.mahafalya Brooks & MichenerL.nilssoni MichenerL.parvula (Darchen)L.vetula Moure & Camargo†L.voeltzkovi (Friese)L.aethiopica Engel†Meliplebeia includes species superficially resembling the smaller Plebeiella and found from Gambia to Eritrea and Somaliland, and southward to Namibia and northern South Africa. Unlike Plebeiella, the basal area of the propodeum is pubescent. Nests are built like those described for Axestotrigona (supra).M.nebulata (Smith)M.beccarii (Gribodo)M.gambiana MoureM.roubiki (Eardley)Meliponula includes a modestly large and robust species, 6–8 mm in length, found commonly throughout tropical Africa, from Guinea eastward to Kenya and thence southward to Namibia and Botswana. The genus is distinctive for the wholly declivitous propodeal basal area, dull and matte metasomal terga, and restriction of the corbicula to less than the distal half of the metatibia. Nests are constructed within pre-existing cavities in trees, and the brood are arranged in irregular layers (The genus r layers  or can ar layers .M.bocandei (Spinola)Plebeiella includes small bees most similar to Meliplebeia but differing in the glabrous basal area to the propodeum. The genus occurs from Togo eastward to Kenya and southward to Angola and Zambia. Nests are built like those described for Axestotrigona (supra).The genus P.griswoldorum (Eardley)P.lendliana (Friese)Plebeia and includes a small species (4–5 mm in length) that can be found from Senegal eastward to Ethiopia and then southward to Angola and northeastern South Africa. Nests are built like those described for Axestotrigona (supra).This genus superficially resembles the New World P.armata (Magretti)Hypotrigonina A.cassiae (Cockerell)A.cincta (Mocsáry)A.essingtoni (Cockerell)A.magna Dollin, Dollin, & RasmussenLiotrigona. Recently, however, the discovery of the male demonstrated that the species was more dramatically different from true Liotrigona than originally surmised. Instead, the type species seems more similar to Austroplebeia and was therefore reclassified Geniotrigona Moure includes large robust stingless bees with a prominent elevated ridge on the vertex posterior to the ocelli, a long malar space, and dense plumose setae on the mesosoma. The genus occurs from Southeast Asia through Malesia. A key to the species was provided by G.lacteifasciata (Cameron)G.thoracica (Smith)Heterotrigona Schwarz includes species similar to Homotrigona Moure, but distinctly smaller in size and much reduced mandibular dentition. Three subgenera  occur west of the Wallace Line, while the remaining two are found exclusively east of the line. Keys to the species of Platytrigona Moure and Sahulotrigona Engel & Rasmussen were provided by Sundatrigona was provided by The genus H.hobbyi (Schwarz)H.bakeri (Cockerell)H.erythrogastra (Cameron)H.itama (Cockerell)H.flaviventris (Friese)H.keyensis (Friese)H.lamingtonia (Cockerell)H.planifrons (Smith)H.paradisaea Engel & RasmussenH.taraxis EngelH.tricholoma EngelH.lieftincki (Sakagami & Inoue)H.moorei (Schwarz)Homotrigona includes those larger species with pronounced mandibular dentition. Some authors have afforded the individual subgenera generic rank H.anamitica (Friese)H.fimbriata (Smith)H.lutea (Bingham)H.canifrons (Smith)H.haematoptera (Cockerell)H.apicalis (Smith)H.binghami (Schwarz)H.melanoleuca (Cockerell)H.peninsularis (Cockerell)H.vidua (Lepeletier)Lepidotrigonanitidiventris (Smith), L.palavanica (Cockerell), L.latipes (Friese)]. Other species have a more typical metatibia with a smaller corbicular surface, such as L.arcifera (Cockerell), while there are those that have a seemingly intermediary form between the extremes . The genus occurs from India eastward to the Philippines and then across Malesia.As the generic name implies, the genus is noteworthy for the presence of abundant, short, decumbent, plumose  setae covering the mesoscutal surface. Additionally, the integument is generally dull and matte, the basal area of the propodeum is reticulate, and the simple setae of the metatibial retromarginal edge. The metatibia and associated corbicular surface, of some species, is greatly expanded, resulting in a spoon-shaped leg, and often associated with an expanded metabasitarsus , the genus to which the species was originally placed. The gender of the name is feminine.The new genus-group name is a combination of the Ancient Greek alpha privative A.aethiopica (Lepeco & Melo), comb. nov.†Meliponini and also the oldest definitive bee.This is an interesting fossil preserved in amber from New Jersey that dates from near the end of the Maastrichtian and is therefore the earliest fossil C.prisca †Trigonisca-like species of Meliponini in Fushun amber, but subsequent studied indicated that the fossil was not in Eocene amber from China but instead likely from the Baltic region. The provenance of the holotype needs considerable study.This genus was described for a E.velteni Engel & Michener†Kelneriapis Sakagami is known only from a single worker preserved in Eocene Baltic amber. Liotrigona and the extinct genus Liotrigonopsis Engel from the same deposits.K.eocenica (Kelner-Pillault)†Liotrigona.This genus, like the two preceding genera, is known only from middle Eocene Baltic amber. Currently, there is only a single worker known. The genus and species were characterized by L.rozeni Engel†th century and so it remains a poorly understood group, but likely belongs to the Hypotrigonina †P.silacea (Wille)†P.tantilla Camargo, Grimaldi, & Pedro†P.vetusta Camargo, Grimaldi, & Pedro†Coccidae), collecting from fungi, and others scavenging the decomposing carcasses of vertebrates , are known as “lambe-olhos”, “lameojos”, or “chupaojos” (lick eye)  tavaresi Camargo & Pedro whereby the bees collected pollen from the surface of flower petals that had been dropped by larger bees (Lestrimelitta and Liotrigona (Cleptotrigona) are robbers that collect their materials from the nests of other meliponines, either by mass invasion, as in the former or by stealthy, spy-like gathering by isolated workers in the latter  . Foraginsurfaces . A similger bees . At an ee latter . Trichotomelitta .Apini) in which it is the old queen that departs to establish the new colony  longula; Nests vary widely but can be generally summarized as follows Fig. : At the Meliponini. Many species build nests within pre-existing cavities in tree trunks or branches, with a preference for spaces that have narrowed openings with the surrounding environment, and which can be easily closed by the bees during nest construction   . Only twCecropia . A diverOxytrigona . If grabbed, many stingless bees will bend the metasoma around to their attacker, mimicking the behavior of stinging, perhaps relying on a Batesian-like mimicry with stinging bees and wasps. Lastly, some species, such as Frieseomelittasilvestrii (Friese), the bees will play dead (thanatosis) when they encounter a large enemy  sting and lack an ability to sting it does not make them defenseless. They exhibit different mechanisms for protecting their nests, ranging from camouflaging the nest entrance, nest construction in places difficult to access, to an active defensive behavior, sometimes quite elaborate. They can tangle themselves in hair and fur, pinching the skin of the aggressor or intruder with their jaws, which can even cause some injuries, enter the nostrils, and ears of intruders, as well as depositing plant resins or caustic substances on the intruder, this last specialized behavior observed with the mandibular glands of bees in the genus ge enemy .


Blood parasite infections such as anaplasmosis, babesiosis, and ehrlichiosis are commonly found in domestic dogs, which adversely influence their health. Many dogs are infected with multiple blood parasites that cause more severe diseases than a single infection. This study aimed to investigate the effect of multiple blood parasite infections on the hematological profiles of dogs at a shelter in Southern Thailand.The blood samples from 122 dogs were collected to assess the hematology profiles of uninfected, single-infected, and multiple blood parasite-infected dogs. The results were compared using Kruskal–Wallis test and Dwass–Steel–Critchlow–Fligner pairwise comparisons. The infections were confirmed by polymerase chain reaction.The results showed that all the infected dogs had significantly lower red blood cell (RBC) count, hemoglobin (HB), hematocrit (HCT), and platelet count (PLT) than the uninfected dogs. Although the dogs with triple infection had lower RBC, HB, HCT, and PLT than those with double and single infections, the difference was not statistically significant.Anaplasma platys, Babesia vogeli, and Ehrlichia canis caused more severe disease than double and single infections. Evaluating the hematological profiles of dogs naturally infected with single, double, and multiple blood parasite infections without clinical signs can enhance their health and welfare.We proposed that triple blood parasite infection with  Babesia vogeli, Ehrlichia canis, Anaplasma platys, Hepatozoon canis, and Trypanosoma spp., regularly observed in dogs in Thailand [Rhipicephalus sanguineus), a common tick species [Ehrlichiosis, babesiosis, anaplasmosis, hepatozoonosis, and trypanosomiasis are serious vector-borne diseases caused by blood parasites in Thailand. Although these diseases are commonly found in animals –6, they Thailand , 9, 10,  species , 12.A. platys rarely cause clinical disease, it can coinfect with other pathogens [B. vogeli and E. canis [Transmission of tick-borne diseases is increasing across Thailand and Southeast Asia , 9–12 anathogens . Microscathogens , 10, espSingle infections with blood parasites affect the health of dogs, causing anemia and, occasionally, thrombocytopenia and leukopenia , 14–20. Therefore, evaluating the hematological profile to detect multiple blood parasite infections in dogs might improve the diagnosis, prognosis, and treatment plan. This study aimed to compare the effects of single, double, and triple blood parasite infections on the hematological profile of naturally infected dogs.This study was approved by the Institutional Animal Care and Use Committee of Walailak University (WU-AICUC-63-036).The blood samples were collected in March 2021 at a dog shelter in Nakhon Si Thammarat province, Thailand. The samples were processed at Small Animal Teaching Hospital Laboratory, Akkhraratchakumari Veterinary College and Research Institute for Health Sciences laboratory, Walailak University.The total dog population in the dog shelter was 487. Blood was collected from approximately 25% of the dogs (122 dogs). The inclusion criteria consisted of healthy dogs without any apparent external clinical signs. Their blood samples were collected from their cephalic veins and transferred into 3 mL ethylenediaminetetraacetic acid (EDTA) tubes for complete blood count and PCR analysis. Approximately 80% of the dogs in the shelter were female (389 from 487 dogs). Most dogs were mongrels and neutered. The shelter was an open-air style with 50% roof covering. The dogs were given adequate food and water. All the dogs were annually vaccinated with combination vaccines  and injected ivermectin regularly to prevent ecto- and endo-parasites.B. vogeli, E. canis, Hepatozoon canis, A. platys, and Trypanosoma spp., were screened using 10% Giemsa-stained thin blood smear under 1000× magnification of a light microscope  and confirmed using conventional PCR. The primers for the detection of blood pathogens were used as previously described [Hematological analysis was performed using an automated machine Procyte Dx . The parameters included red blood cell (RBC), hemoglobin (HB), hematocrit (HCT), mean corpuscular volume, mean corpuscular HB, mean corpuscular hemoglobin concentration (MCHC), RBC distribution width (RDW), white blood cell (WBC), neutrophil, lymphocyte, eosinophil, monocyte, and platelet counts (PLTs). Five blood parasite species, including escribed –30.® Blood DNA Kit  according to the manufacturer’s instructions, and its concentration was measured using the Nano-Drop™ spectrophotometer . The PCR reaction mix contained 6.25 μL DreamTaq Green Master Mix (2×) , 1–2 μL DNA template (100–200 ng/uL), 0.5 μL primer (0.4 μM) and nuclease-free water to a final volume of 12.5 μL, and was conducted using the Mastercycler Pro S machine . The cycling conditions were as follows: Initial denaturation step at 95°C for 3 min, denaturation at 95°C for 30 s, and annealing at 54°C  or 58°C (for A. platys and Trypanosoma spp.) for 30 s, extension at 72°C for 1 min for 35 cycles; and a final extension at 72°C for 5 min. The positive control included the DNA of known blood parasites from our previous study [For PCR analysis, DNA was extracted using the E.Z.N.A.The data were analyzed using Jamovi 2.3.9 software . The data were represented as mean ± standard deviation. The hematological parameters were compared and statistically analyzed using the Kruskal–Wallis test and Dwass–Steel–Critchlow–Fligner pairwise comparisons. p < 0.05 was considered statistically significant.A. platys, E. canis, or B. vogeli .Of the 122 dogs tested, 58 were uninfected, while the remaining  were infected with at least one of these parasites; . vogeli . None ofAnaplasma platys-infected dogs had the lowest HB and HCT, while the RBC, HB, and HCT values of E. canis-infected dogs were significantly lower than those of the uninfected dogs  than in the uninfected dogs. The neutrophil count of E. canis-infected dogs was significantly lower than that of uninfected dogs. The lymphocyte counts were higher in the E. canis- and A. platys-infected dogs than the uninfected ones but within the normal range. The highest lymphocyte count was seen in A. platys-infected dogs without any significant difference. The eosinophil count was higher in the A. platys-infected dogs than the uninfected dogs but within the normal range. The monocyte counts were highest in E. canis-infected dogs but within the normal value   and thrombocytopenia decreased from multiple infections to single and non-infection, respectively . We did < 0.001) .There was no significant difference in the WBC count in all the groups. Neutrophil count in single infection was the lowest among all infection groups, which was significantly lower than in uninfected dogs. Higher lymphocyte counts were observed in single, double, or triple infections without any significant difference. Monocyte counts in triple infection and eosinophil counts in single infection were lower than in the other groups, without any significant difference.We observed that canine anaplasmosis is associated with anemia in Thailand , 18, unlE. canis infection mainly causes leukopenia in Bangkok and Kanjanburi [E. canis-infected dogs, which were over 50% lower than that reported previously from a neighboring area [Consistent with the previous studies, we showed that njanburi , 24, 31 njanburi . We alsoing area  but wereing area .B. vogeli in Songkhla, Bangkok, and Kanchanaburi [B. vogeli infections through randomized sampling. None of the dogs showed clear signs of babesiosis.Lymphopenia was observed in dogs infected with hanaburi , 20, butB. vogeli and E. canis exacerbates the disease, which showed lower RBC, HB, HCT, and lymphocyte counts than single B. vogeli infection [A. platys and E. canis displayed lower HCT and PLTs than single-infected ones [Two reports from Thailand with different types of co-infection data showed that multiple infections significantly reduce the RBC, HB, HCT, and PLTs , 24. Anonfection . A previted ones . As blooted ones , 24, 31.Babesia rossi displayed significantly lower mean lymphocyte count and percentages of CD3+, CD4+, and CD8+ T lymphocytes than uninfected dogs [B. vogeli-infected dogs.Blood parasite-infected dogs showed lower lymphocyte count. Canine monocytic ehrlichiosis (CME) showed a higher correlation with T- and cytotoxic T (Tc) cells, and the absolute number of Tc cells was higher in peripheral blood. The percentage of T helper cells and the absolute and relative values of B lymphocytes were higher in healthy dogs than in CME dogs . Dogs nated dogs . Here, wet al., unpublished data).In this study, the infected dogs did not show obvious clinical signs of blood parasite infection by physical examination. However, their hematological analysis indicated significant anemia and thrombocytopenia. Treatment of multiple blood pathogen infections in dogs is more complicated and takes longer than a single infection. Our preliminary study data of multiple blood parasite infection treatment in dogs appear to be improved with the hematology profile and devoid of parasites in blood circulation when given 10 mg/kg doxycycline once a day for at least 8 weeks (Boonhoh A. platys and B. vogeli co-infection and multiple infections with H. canis and Trypanosoma spp. Few A. platys and B. vogeli, single infection cases were detected in this study, which might not adequately represent the hematological profile of single blood parasite infection with A. platys and B. vogeli in dogs.Our study had some limitations. Due to the limited number of dogs, randomized group sampling, time of sampling, and limited sample collection location, we could not explore the hematological data of A. platys, B. vogeli, and E. canis cause more severe disease than double and single infections. Further treatment guidelines for multiple blood parasite infections in dogs should be investigated to enhance the health and welfare of these animals.There are limited studies on multiple blood pathogen infections in dogs. Based on the hematological profile of the tested dogs, we proposed that triple multiple blood parasite infections with TW: Designed and supervised the study, participated in sample collection, and revised the manuscript. WB: Performed physical examinations, collected blood samples, analyzed data, and prepared and revised the manuscript. NS: Performed PCR and participated in sample collection, data analysis, and revised manuscript. PF: Participated in sample collection and data analysis. All authors have read, reviewed, and approved the final manuscript.