Nanopore sensors provide a unique platform to detect individual nucleic acids, proteins, and other biomolecules without the need for fluorescent labeling or chemical modifications. Solid-state nanopores offer the potential to integrate nanopore sensing with other technologies such as field-effect transistors (FETs), optics, plasmonics, and microfluidics, thereby attracting attention to the development of commercial instruments for diagnostics and healthcare applications. Stable nanopores with ideal dimensions are particularly critical for nanopore sensors to be integrated into other sensing devices and provide a high signal-to-noise ratio. Nanopore fabrication, although having benefited largely from the development of sophisticated nanofabrication techniques, remains a challenge in terms of cost, time consumption and accessibility. One of the latest developed methods—controlled breakdown (CBD)—has made the nanopore technique broadly accessible, boosting the use of nanopore sensing in both fundamental research and biomedical applications. Many works have been developed to improve the efficiency and robustness of pore formation by CBD. However, nanopores formed by traditional CBD are randomly positioned in the membrane. To expand nanopore sensing to a wider biomedical application, controlling the localization of nanopores formed by CBD is essential. This article reviews the recent strategies to control the location of nanopores formed by CBD. We discuss the fundamental mechanism and the efforts of different approaches to confine the region of nanopore formation. I, connects to the volume of the electrolyte solution displaced by the particle, thereby revealing the size of the target analyte. The duration of the current drop, or dwell time (dt), corresponds to the diffusion coefficient of the particle, providing information about the charge and size of the particle. With rigorous analysis, one can obtain information related to the particle shape, volume, charge, dipole moment, and diffusion coefficient [Nanopore sensing has been attracting attention rapidly over the past decades due to its appealing applications in label-free, real-time biosensing of single molecules in aqueous solutions ,3,4,5,6.fficient ,18,19,20fficient ,27,28,29mycobacterium smegmatis porin A (MspA) [aeromonas hydrophila Aerolysin (AeL) [bacteriophage phi29 (Phi29) [There are two major categories of nanopores—biological nanopores and synthetic, solid-state nanopores. Biological nanopores consist of single transmembrane proteins inserted in lipid bilayers or polymer membranes. Commonly used biological pores, such as α-hemolysin (αHL) ,33,34,35A (MspA) ,37,38, ain (AeL) ,40, and  Phi29) ,42, have (Phi29) ,41,42, p (Phi29) ,44, and  (Phi29) . Moreove9 ,42, ha (Phi29) . Since 2 (Phi29) , and the (Phi29) ,47.Despite these attractive advantages, biological nanopores suffer from several drawbacks ,6. First2 [2 [On the other hand, the diameter and geometry of synthetic nanopores can be tuned depending on the user’s requirement. These nanopores can be fabricated in a wide range of materials including silicon nitride ,54,55,562 , and pol2 , as well2 ,61, MoS22 [2 , and BN 2 [2 . The inh2 [2 ,68,69,702 [2 ,67,71,72Recent developments in nanofabrication allow a wide variety of approaches to fabricate such pores, including but not limited to transmission electron microscopy (TEM) drilling ,49,50, fDue to the barriers to fabricating nanopores mentioned above, the controlled breakdown (CBD) method of nanopore fabrication immediately attracted wide attention since it was reported in 2014 by the Tabard-Cossa group . CBD useDespite of all these advantages, CBD nanopore fabrication comes with some disadvantages in comparison to other fabrication techniques. Firstly, multiple nanopores can be formed by CBD. During the past five years, several works have attempted to reduce the formation of multiple nanopores by different approaches ,90,91,92In recent years, combining nanopore sensing with fluorescence and plasmonic optical measurements has drawn more and more interest, because optical signals provide additional depth of information without interfering with the electrical signal ,107,108.When a dielectric material is under high electric field stress for a certain duration, the material loses its insulating properties locally. This process, or the failure of dielectric materials under electric field stress, is known as dielectric breakdown ,114. Mosx membrane. An external electric field across the membrane, typically 0.6–1 V/nm, induces defects inside the membrane due to electric field stress. These defects increase the leakage current tunnelling through the dielectric membrane and accelerate the accumulation of defects . When the density of defects is high enough to form a connecting path in the insulating membrane, the current tunnelling through this path locally removes the membrane material in a short time, creating an open channel inside the membrane. Nanopores with an initial size distribution from 0.5 to 3 nm can be formed if the external electric field is removed immediately after the sudden increase of current. This process is the fabrication phase of CBD, and only one nanopore is expected to form during this phase.Similar to the breakdown of thin films in a dry atmosphere, breakdown can occur in a liquid environment when a thin membrane is under external electric field stress. Instead of aiming to avoid breakdown, Tabard-Cossa’s group took advantage of this stochastic process for nanopore fabrication, which is now widely known as controlled breakdown (CBD) . Figure For protein characterization, there is a need for a vast variety of nanopore sizes, ranging from several nanometers to one hundred nanometers. After the initial breakdown, further applying an electric field with feedback control allows for the expansion of and precise control over the nanopore size. During this conditioning phase of CBD a, the apx membranes with diameters ranging from single nanometers to several tens of nanometers [x membrane on a fused-silica substrate, because the charged particles used for drilling cannot be conducted away. CBD, on the other hand, can easily form nanopores in this substrate, which enables the production of low-noise nanopore devices [2 [2 [2 [CBD has been rapidly applied in fabricating nanopores within different materials and in different sizes. nometers ,91,118.  devices . CBD hasvices [2 , HfO2 [5es [2 [2 , and metes [2 [2 ,121, as [2 [2 [2 ,128,129.Despite its many applications, fabricating nanopores by CBD comes with drawbacks due to the intrinsically random nature of the breakdown process. The issues of the CBD technique that need to be addressed include: (1) the possibility of a long and uncertain amount of time necessary to fabricate a nanopore; (2) false breakdown events due to soft breakdown; (3) the potential to form multiple nanopores in the membrane that are difficult to identify using electric current feedback; (4) pore formation in random locations in the membrane; and (5) the impossibility of using CBD to form nanopore arrays.An ideal controlled breakdown protocol would meet the following criteria to allow researchers to benefit from its low cost and broad accessibility: (1) a short time-to-breakdown to allow efficient pore fabrication; (2) precise size control of the generated nanopore, including both a tight and small size distribution of initial nanopores in the fabrication phase, as well as an efficient feedback control over the pore size during the conditioning phase; (3) the production of single nanopores in the membrane rather than forming multiple nanopores; (4) a smooth surface and regular nanopore shape to allow a high signal-to-noise ratio for nanopore sensing; and (5) control over the localization of nanopores formed in the membrane.x membranes with different thicknesses by applying a high constant electric field while measuring the time-to-breakdown [In the context of dry membranes, the time-to-dielectric breakdown is strongly dependent on the parameters of the material used, including the thickness of the membrane, the composition of the material, and the thin-film deposition process. In addition to the intrinsic nature of the materials, several external conditions also contribute to the time-to-breakdown of the thin membrane, including the electric field strength, external induced defects, and temperature. Tabard-Cossa’s group analyzed the stochastic breakdown process of SiNreakdown . The disreakdown . Thus, tHere, we discuss the parameters influencing the breakdown in both dry and liquid conditions, as well as the parameters that only apply to the liquid environment, such as the pH and salt concentration of the electrolyte.x thin film affects the breakdown process strongly due to the changes in electrical conductivity associated with the Si content [x is often used for nanopore sensing due to its excellent mechanical stability, allowing it to be fabricated into large-area free-standing membranes [x membranes is favorable for CBD fabrication, because high silicon to nitride ratios lead to a high density of charge traps [x membranes tend to expand and grow over time when immersed in a salt solution [x membranes when voltage is applied via electrolyte solution, electrodes, and the combination of both. For Si-rich SiNx membranes, a high voltage is required to induce breakdown, because the electrochemical reaction between the electrolyte and dielectric interface limits the conduction across the membrane.Research on thin membranes of electronic devices shows that different materials exhibit different breakdown properties ,133,134, content ,131,132. content ,91,120. embranes ,136. Thege traps ,91,120. solution . Recentlsolution  studied x, CBD has been used for fabricating nanopores in other insulating materials such as SiO2 [2 [2, SiNx, and HfO2 by investigating their breakdown potential using linear sweep voltammetry [x is not sensitive to cations such as sodium or silver ions. These ions, however, have a significant influence on the breakdown potential of SiO2 membranes. HfO2, on the other hand, is resistant to sodium ions but strongly affected by silver ions, likely due to the intrinsic oxygen vacancies. In addition to pure insulating materials, CBD has demonstrated its ability to generate nanopores in metal-insulating layers [In addition to the SiN as SiO2  and HfO2 SiO2 [2 . Yun et tammetry . They fog layers ,121. Thig layers , which ix membranes with a thickness between 10 to 30 nm with a breakdown field strength typically between 0.6 to 1 V/nm [Most nanopores fabricated by CBD are in SiNo 1 V/nm ,132,139.o 1 V/nm . Beyond o 1 V/nm , which io 1 V/nm ,140.In addition to the above parameters, liquid-contacting membranes have some unique parameters that also affect the breakdown process. When the solid material is in contact with liquid, the electrostatic field on the surface of the solid material attracts the counterions in the liquid, forming an electrical double layer at the solid–liquid interface. When there is a potential difference across the interface, electrochemical reactions occur at the interface, which induces the injection and/or removal of charges in the material . The intOther parameters, such as solute concentration and solvent type, have also been shown to affect the breakdown process ,138,141.There are two feedback controls required in CBD nanopore fabrication. One is the detection of the breakdown, and the other is the termination of the external electric field during nanopore enlargement. Depending on the applications, different approaches are used and/or combined with feedback control to optimize nanopore fabrication by CBD.Detection of a breakdown event. An efficient feedback control system allows a high yield rate of nanopore fabrication and a tight size distribution of initial nanopores. The simplest feedback control is to use a cut-off threshold when applying constant electric field stress [d stress ,142. Thid stress ,90.To differentiate a soft breakdown from a hard breakdown, Roshan et al.  developeInstead of applying a constant electric field across the membrane, other protocols use ramping voltage pulses or constant currents to create an external electric field stress. The strategy of ramping the voltage can be very useful and efficient for membranes from different batches, e.g., batches with different thicknesses or material compositions ,83,140. Termination of the breakdown process when the pore meets the desired size. After the detection of the initial breakdown, a CBD protocol usually reduces the applied electric field to slowly expand the nanopore to the desired size. To estimate the pore size during the enlargement process, one can measure the resistance across the membrane by measuring one of the current, the voltage, or the IV curve during the breakdown. The accuracy of the pore size and the time of the feedback are two key features to ensure the size control of nanopores.This conditioning phase carries the risk of potentially generating multiple nanopores in a membrane ,94,143; Several approaches have been proposed to monitor the nanopore enlargement process in addition to the electrical signal feedback. Measuring the conductivity change after coating the membrane with a self-assembled layer can report whether a single pore is formed in the membrane ,122. Thi2+ ions and Ca2+ indicator [On the other hand, optical signals can report the exact number of pores formed in a membrane. Optical measurements also have no interference with the electrical signal and hence can be implanted in a system as an orthogonal source of information. Three different optical signals have been explored for monitoring nanopore formation during the breakdown in real time: (1) photoluminescence (PL) intensity ,89,145, ndicator ,88,90. WAll optical measurements as feedback controls come inherently with low spatial resolution due to the optical diffraction limit. This resolution cannot discriminate multiple nanopores that are formed in the focus area, e.g., Figure 4 of Ref. . In addiOptical nanopores  can beneOne year after CBD was reported, the Dekker group first reported a self-aligned nanopore formation at an optical hotspot using laser-induced breakdown . They dex membrane with a spot size of a micrometer or less. An additional optical feedback control  laser-accelerated defect accumulation, (2) laser thinning, and (3) thermal-assisted etching/breakdown. Although the fundamentals of laser-assisted breakdown can vary between experiments, the setup of laser-assisted breakdown remains similar. Whether we detect a thinned membrane or defect accumulation of the membrane depends on which fundamental mechanism governs the pore-formation process. The governing mechanism for accelerated pore formation with the assistance of laser illumination depends on the laser wavelength, the material of the membrane and its surrounding environment. Through different characteristic approaches, one can figure out which mechanism governs pore formation.Laser-accelerated defect accumulation. When a laser illuminates the dielectric membrane, it promotes the rate of defect accumulation and hence induces a higher leakage current [ current . Such la current . These d current . Both thLaser thinning. The laser etching of a SiNx membrane is a well-established technique for nanopore fabrication. This approach relies on the photochemical destabilization of Si–Si bonds in a Si-rich SiNx membrane. Combining CBD setup with laser thinning provides in-situ nanopore formation, which can be beneficial for integrating the nanopore with microfluidics and biosensing , as shown in x membrane [d et al. ). Duringolutions ,87,158. olutions ,145,159.membrane .Thermal-assisted etching/breakdown. Both laser-induced defect accumulation and laser-thinning processes are accompanied by a temperature increase due to laser heating. When the nanopore is illuminated by a laser beam, the membrane absorbs the laser radiation and converts it to joule heating. This localized laser-induced heating accelerates the reaction rate of either the breakdown process or the thinning process. In addition to its role in thermal-assisted etching/breakdown, laser heating has been used for nanopore fabrication, e.g., the photon-to-heat graphene nanopore sculpting [culpting  and the culpting ,162.Electron and light microscopy images indicate an irreversible change in the membrane material after laser illumination, either due to laser-induced defect cumulation or laser thinning. The dominant mechanism varies among experimental conditions, e.g., the laser wavelength, applied voltage, and electrolyte solution. Generally, laser thinning occurs when the membrane is illuminated by a blue or green laser (488 or 532 nm), while a near-infrared laser normally introduces defect cumulation inside the membrane. The dominant mechanism for pore formation can be characterized by transmission light microscopy—a laser-thinning region appears bright while laser-induced defect accumulation appears dark, as shown in In addition to the localized pore formation, laser-assisted breakdown also allows the fabrication of nanopore arrays ,90,159 aThe integration of an optical setup with CBD provides a unique platform to characterize nanopores formed by CBD in real time. As discussed in Photoluminescence (PL) intensity. The photoluminescence (PL) is the light emission from materials [x membrane covers a spectral range from UV to IR [aterials . The PL UV to IR ,176,177.UV to IR ,89. WhenUV to IR ,159.Measuring ionic current by laser scanning. This approach relies on the increased ionic conductivity of the electrolyte due to local heating when a laser is focused on the nanopore location. The membrane, often silicon nitride, absorbs the partial laser energy and converts it to heat. This heat cannot disperse well due to the boundary between the membrane and the liquid, resulting in local temperature enhancement. x membrane increases the temperature of the electrolyte in the nanopore. As the temperature increases, the viscosity of the electrolyte decreases [ecreases ,190,191,ecreases . Therefoecreases ,95. Sincecreases ,95,145.2+ flux detection.Ca Monitoring the Ca2+ concentration near the nanopore by using a Ca2+ indicator provides an optical signal to detect ion flux through a nanopore [2+ indicator dye (Fluo-8 or Fluo-4) changing in response to the concentration of Ca2+ ions [2+-indicator increases when it binds to the freely diffusing Ca2+ ions, providing information about the local concentration of Ca2+ ions near the nanopore. 2+ nanopore detection method. Ca2+ ions and Ca2+ indicator dyes are separated by the membrane with the nanopore as the only connection. When the applied voltage is negative, the electrophoretic force pulls Ca2+ ions by diffusion from the trans to cis chambers through the nanopore. When the polarity is reversed, the Ca2+ is driven from cis to trans through the nanopore and binds to the Ca2+ indicator dyes on the other side of the reservoir. The Ca2+ indicator then emits fluorescence that is localized near the entrance of the nanopore. Monitoring the Ca2+ ion flux through nanopores enables an optical readout to provide real-time, widefield feedback of the quantity of nanopores formed in the membrane [nanopore ,193,194.a2+ ions . The flumembrane ,90. ThisIn addition to the above methods, tip-induced breakdown offers another way to generate nanopores locally in a membrane and reduces the possibility of forming multiple nanopores. This group of methods relies on pre-defining a small region with either HIM, an AFM tip, or a micropipette. This small region limits the area of pore formation to a much smaller space than the whole free-standing membrane.2 on a low-stress SiNx membrane with a window size of 100 × 100 µm2 . This method offers the most precise position control of any currently known CBD technique and largely reduces the possibility of forming multiple nanopores. The use of AFM, however, increases the cost of pore fabrication, and the alignment processes can be time consuming.Zhang et al.  further Similarly, liquid meniscus contact using a glass pipette can also form nanopores in a precise position . As illuIn addition to using tip-based confinement, the electric field can also be confined by electrolytes within microfluidic systems to fabricate nanopores precisely, as illustrated in 2O: C2H5OH (1:8:3) solution (cis) and NaCl solution (9 mg/mL) (trans), respectively. The high external electric field accelerates the generation of holes (h+) in silicon, which collides with the crystal lattice to free the bound electrons. When the current density is high enough, the following electrochemical reactions occur:Linaro’s group combined chemical etching with CBD to fabricate high aspect ratio silicon nanopore arrays . They deAs illustrated in x membrane [Very recently, Fried et al. demonstrated the pore formation locally at the area of metal electrodes by locally injecting electrons into the membrane. They deposited metal electrodes on the surface of the SiNmembrane . These mmembrane  have alsSince the nanopore-sensing concept was proposed 20 years ago, the nanopore technique is now at the stage of moving toward real-world applications and marketing. One main challenge to this progress, however, remains the fabrication of ideal nanopores with precisely controlled sizes, shapes, and locations. Controlled breakdown (CBD) has greatly improved the nanopore-sensing field by providing a low-cost and broadly accessible approach. Localized nanopore fabrication via CBD will further push the nanopore technique to the broader biosensing community. Future development of CBD techniques might focus on addressing questions/obstacles of CBD techniques such as the breakdown mechanism of different membrane materials, improving the long-term stability of CBD nanopores, and minimizing the surface roughness of the pore channel. All the above aspects are crucial for CBD techniques to be integrated into different nanodevices. Combining CBD with advanced techniques, such as nanofluidics, nanophotonics, AFM, or field transistors with 2D materials, will allow nanopore sensing to move closer to being a ready-to-use tool on the market, not only in DNA sequencing, but also in monitoring environmental pollution and ultra-sensitive biomarker detection. We believe that, in the future, the fields of biomedical and bioengineering, biosensing, nanofluidics, and integrated sensing devices will benefit significantly from the nanopore technique.


Oedogonium, Oedocladium, and Bulbochaete, which include more than 600 described species. The classification of Oedogoniaceae is currently based on morphology, and the complicated morphological characteristics make species identification difficult, with the limited molecular data also restricting the phylogenetic analysis. In the present study, we collected 47 Oedogonium specimens from China and sequenced 18S rDNA, ITS2, ITS (ITS1 + 5.8S + ITS2), and rbcL sequences to conduct phylogenetic analyses. We selected nine morphological characteristics, most of which were considered important in traditional systematics, for comparison with the molecular phylogeny results. All the topologies based on different datasets showed similar results; Oedogonium was a paraphyletic group, and Oedocladium and Bulbochaete clustered with Oedogonium. The morphological characteristics matching the phylogenetic results showed that the types of sexual differentiation, characteristics of the oogonium , division types of antheridial, and number of sperm of each antheridial, which are considered the most important morphological characteristics in traditional taxonomy of Oedogonium, did not form monophyletic lineages respectively, indicating that traditional systematics may not reflect the real phylogeny of the genus Oedogonium. In addition, a new taxonomical classification of the genus Oedogonium was presented according to the shapes of basal cells, which matched well with the phylogenetic topologies. In addition, we propose to divide the genus Oedogonium into two sections, section Globosum and section Elongatum, representing the species with spherical or sub-hemispherical basal cells and elongated basal cells, respectively.Oedogoniales comprises the three genera  Oedogonium Link ex Hirn, Oedocladium Stahl, and Bulbochaete Agardh [Oedogonium includes 444 species and 349 lower taxonomical units, genus Bulbochaete includes 113 species and lower taxonomical units, and genus Oedocladium includes 15 species [Oedogonium has simple unbranched filaments; Oedocladium has branched filaments; and Bulbochaete forms bulb-based hairs [The order Oedogoniales belonging to the family Oedogoniaceae Chlorophyceae, Chlorophyta, includes three genera, e Agardh ,2,3,4. Med hairs ,11,12,13ed hairs ,15,16.Oedogonium. Sexual differentiation could be the criterion for taxonomical classification for all species of Oedogonium, according to the position and relation of the oogonium and the antheridia could be distinguished as monoecious; dioecious, macrandrous; dioecious, and nannandrous [Oedogonium. Mrozińska [Oedogonium.During the past few years, phycologists have used different characteristics as criteria for dividing the genus nandrous ,10. Gautrozińska ,18 propoBulbochaete may be a sister to the other two genera [Oedogonium species, Alberghina et al. [Oedogonium might not be monophyletic and that the morphological characteristics may not define the phylogenetic groups. Using 18S rDNA sequences, Mei et al. [Oedocladium formed a separate clade within Oedogonium, whereas Bulbochaete was relatively distant from the other two genera. Xiong et al. [Oedogonium was paraphyletic based on chloroplast genome protein-coding genes [With the development of molecular phylogenetics, phycologists later conducted a series of phylogenetic studies of Oedogoniales. Oedogoniales are monophyletic, and o genera ,20,21,22a et al.  suggestei et al.  found thg et al. ,26,27,28ng genes ,26, mitoOedogonium has described a large amount of species based on morphology, the molecular phylogenetic studies included relatively limited samples, the molecular data in the public database about the order Oedogoniales are also limited, and the phylogeny of Oedogonium remains problematic. In addition, the relationships that the morphological characteristics used as criterion of Oedogonium with the molecular phylogenetic result have not been discussed comprehensively; more molecular data and a broader sampling is required for further study of this group.Even though the genus Oedogonium specimens from China were sampled and the 18S rDNA, ITS, and nucleotide sequences of the chloroplast genome (cpDNA) rbcL gene of the majority of them were obtained for phylogenetic analyses. The relationships between the different morphological characteristics and phylogenetic results were comprehensively discussed. The objective of the present study was to explore the phylogenetic relationship of the genus Oedogonium based on additional taxa and to search for the morphological characteristics which could reflect the phylogeny of Oedogonium.In the present study, a total number of 47 Oedogonium taxa included in this study are listed in Characteristics of the Oedogonium taxa newly sampled together with the sequences downloaded from the NCBI database. The ITS dataset consisted of 375 bp, of which 42.9% was variable informative and 32.5% was parsimony informative. The ITS2 sequence consisted of 213 bp, of which 67.61% was variable informative and 54.00% was parsimony informative. The concatenated dataset consisted of 3159 bp, of which 17.6% was variable informative and 9.5% was parsimony informative. The phylogenetic trees based on ITS sequences by both ML and BI showed the same results: separation of Oedogoniales into two clades with high support value  , ITS2, and 18S + ITS (ITS1 + 5.8S + ITS2) + rbcL of 47 ctively) . In the tic tree , and then clades . In the Oedogonium  with a strong supported value. In the first clade, the taxa possessed elongated basal cells, and in the second clade, they possessed spherical or sub-hemispherical basal cells  were selected to match the phylogenetic tree based on ITS sequences to search for the characteristic reflecting the phylogeny of dogonium . Our resal cells . Hence, Globosum sect.The basal cell spherical or sub-hemispherical; the filaments are relatively small with the width almost less than 10 um; the oogonium operculate, most division median, smooth oospore wall. Oedogonium capitellatum Wittrock ex Hirn . Type species: Elongatum sect.Oedogonium . The basal cell is elongated, and the other characteristics are the same as the description of genus Oedogonium grande Kützing ex Hirn .Type species: Oedogonium was polyphyletic, and Oedocladium was separated into two clades clustering with Oedogonium, which were identified with our previous studies [Bulbochaete and Oedocladium clustered with Oedogonium. Oedocladium is not considered an independent lineage; this was explained by studies by Liu et al. [Oedogonium pakistanense, one of the few terrestrial species belonging to the genus Oedogonium, showed apical growth considered as a typical characteristic of Oedocladium. The authors proposed that Oedogonium pakistanense represents an evolutionary transition between Oedogonium and Oedocladium [Oedogonium, apical growth also occurs in many aquatic species, which showed a cladogenetic pattern and obscured phylogenetic signals between the two genera. Even though exact topologies differed based on ITS, ITS2 and 18 S + ITS + rbcL, they showed a basal clade including identical taxa .In the present study, the phylogenetic results based on ITS, ITS2 and 18 S + ITS + rbcL by both BI and ML methods showed that  studies ,26,27,28u et al. , in whicocladium . Based oOedogonium did not form monophyletic lineages, respectively, implying that the traditional morphological character used for the taxonomy of Oedogoniales may not reflect the real phylogeny of this group. The phenomenon of different types of sexual differentiation evolving multiple times has also been reported in bryophytes and plants [Oedogonium pringsheimii presented with obtuse or apiculate terminal cells in the field or in culture. However, basal cell shapes were found to match well with both of the topologies based on ITS, ITS2, and 18 S + ITS + rbcL, suggesting that basal cell shape is more consistent with the molecular phylogeny results. The basal cell of the filament of Oedogonium is most frequently elongated, and the attached lower end may be simple or lobed. In addition, the developing basal cell of the other Oedogonium species may be flattened into a subhemispherical cell or rarely appear nearly spherical. Based on samples of Jao [Oedogonium included about 440 species, and the number of the species with subhemispherical or nearly spherical basal cells was 33. According to the statistics of the morphological characteristics of these 33 species, we found that they included the three kinds of sexual differentiation types, which showed again that the sexual differentiation types could not reflect the real phylogeny of the genus Oedogonium. In addition, all the 33 species also showed some common characteristics; for example, the filaments are relatively small, with the width almost less than 10 um; and with the oogonium subdepressed-globose; circumcision; and smooth ospore wall, and these characteristics were considered more primitive in the studies based on phenetic- and cladistics taxonomy methods by Mrozińska [Phylogenetic analysis combined with morphological studies revealed the morphological characters  important in the traditional taxonomy of d plants ,35,36,37d plants  and our s of Jao  collecterozińska ,18, implThe 47 specimen strains described in this study were isolated from water samples and deposited in the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB collection), Wuhan, Hubei province, China. Voucher numbers are shown in 2·s). Once a unialgal culture was obtained, total genomic DNA was extracted using a Universal DNA Isolation Kit  according to the manufacturer’s instructions. Polymerase chain reaction (PCR) amplification of partial 18 S rDNA, whole ITS rDNA regions, and rbcL were performed using 5 µL template DNA, 0.1 µM of each primer, and 25 µL 2× Tap Master Mix  in a 50 µL reaction volume. Nuclear-encoded 18 S rDNA sequences were amplified using the primers 18 F (5′-TGGTTGATCCTGCCAGT-3′) and 18 R  once or twice. All strains were grown at 20–25 °C in solid BG11 medium under a 12 h-12 h light-dark cycle at an intensity of 15–30 μmol/. The amCACC-3′;  and depoThe nuclear-encoded 18 S rDNA and ITS (ITS1 + 5.8 S + ITS2) sequences, and nucleotide sequence of the cpDNA rbcL gene were used for phylogenetic analyses. Sequences of related species were obtained from GenBank . In addi


Alliaceae as a potential option due to their variety of bioactive compounds. In this study, we analyzed the antibacterial and antiparasitic potential of propyl-propane-thiosulfinate (PTS) and propyl-propane-thiosulfonate (PTSO) from onions. The in vitro activity against Pseudomonas anguilliseptica, Tenacibaculum maritimum, and Photobacterium damselae of both compounds was tested. In addition, the viability of Sparicotyle chrysophrii larvae was evaluated. Moreover, a diet that consisted of a blend of PTS/PTSO (ALLIUM) was used. A total of 90 gilthead sea bream juveniles were tested against P. damselae subsp. Piscicida after 12 weeks of dietary administration. Furthermore, 150 fish with a rate of 10–15 parasites/fish were fed for 21 days and the number of gill parasites was recorded. All strains were sensitive to both compounds. PTSO showed the highest inhibitory effect against all target strains, while PTS showed higher effectiveness against S. chrysophrii. Fish from ALLIUM group presented the highest probability of survival, increasing up to 91.1%, whereas in the control group, the probability of survival was 66.7%. The number of parasites in the gilthead sea bream decreased in the ALLIUM group over time. These results suggest the inclusion of PTS and PTSO in feed as a natural strategy to prevent antibacterial and antiparasitic fish diseases.The use of phytogenic extracts is considered a sustainable strategy for the prevention of fish diseases, including  Sparus aurata) is one of the most important farmed species in the Mediterranean, increasing steadily from around 96,718 tons in 2004 to an estimated 258,753 tons in 2019 and valued at USD 1.28 billion worldwide [The implementation of intensive and semi-intensive production methods, coupled with the diversity of farmed species and the decline in ocean fish stocks, has positioned aquaculture as the fastest-growing food production sector ,2. AmongPhotobacterium damselae subsp. Piscicida, a Gram-negative bacterium that causes significant production losses due to its ability to infect a wide range of fish species with high mortality rates from lesions, hematological disorders, and histological abnormalities, among other clinical signs [Sparicotyle chrysophrii, a common monogenean parasite that is often responsible for large commercial losses in fish farming and causes lethal diseases through anemia, histopathological damage, hemorrhages and necrosis [One of the most important bacterial infections is photobacteriosis/pasteurellosis, caused by al signs ,5. Gilthnecrosis ,7. Thesenecrosis  highlighTo continue with further sustainable development of the aquaculture industry, several alternative biocontrol methods have been proposed, including good management practices, such as reducing animal density, vaccinations, and phage therapy, together with nutritional interventions that comprise the use of functional additivess, such as probiotics, prebiotics, symbiotics, and phytogenics ,10,11,12Plants contain numerous secondary metabolites that play functional roles, such as protecting plants from herbivores, insects, microbial infections and other challenges, with antimicrobial, anti-inflammatory, antioxidant, and antiparasitic properties, the reason for which their use as traditional medicinal plants has widely expanded . These bAllium spp., such as garlic (Allium sativum) and onion (Allium cepa), hold great potential due to their variety of bioactive compounds, including polyphenols and organosulfur compounds, among others, being one of the most studied plants of therapeutic importance [Alliaceae are known to exhibit antioxidant, antimicrobial, antifungal, and antiparasitic properties, and have also been reported to modulate immune response and upgrade mucosal epithelial barrier functions [Eimeria acervulina in broilers [Among the different phytogenic plant extracts, those derived from portance . In aquaunctions . The mosunctions . Both orunctions , togetheunctions . Moreoveunctions . In terrunctions ,25,26,27broilers . AlthougCaligus rogercresseyi) and fish nematodes, such as Anisakis and Hysterotylacium aduncum [Sparicotyle chrysophrii, a gilt parasite that belongs to the monogenean class, specific to S. aurata, causes high mortality with several financial impacts and whose prevalence and virulence is increasing in warmer seawater due to climate change [In previous studies, we have reported the antiparasitic effects of PTSO against salmon sea lice  studied with P. damselae and S. chrysophrii was also evaluated.Considering this background information, in the present study, the in vitro antibacterial and antiparasitic effects of PTS and PTSO were studied. Moreover, the effect of both compounds in juvenile gilthead sea bream , while those exposed to PTSO did so from a compound concentration of 1 µg/mL upwards (p < 0.01)  . At the p < 0.01) compared with those fed the basal diet   .Alliaceae [The antimicrobial potential of the phytogenic molecules present in medicinal plants has been widely described in the literature, especially those compounds derived from lliaceae ,32. Indelliaceae ,34. ThisP. anguilliseptica, T. maritimum and P. damselae. These results were similar to those obtained by Guo et al., who reported antibacterial activity of garlic powder against P. damselae subsp. piscicida, although obtaining lower inhibition zones and higher MCB values than those found in our in vitro tests with PTSO [Alliaceae, other bioactive compounds have been shown to exert activity against these bacteria. Isothiocyanates from horseradish (Armoracia rusticana) were also able to inhibit P. anguilliseptica, T. maritimum and P. damselae, among others, although, once more, obtaining smaller inhibition halos compared to PTSO [Origanum vulgare also exerted antibacterial activity against P. damselae [Our results showed that PTS and PTSO were able to inhibit ith PTSO . Similar to PTSO . Neverthdamselae .It is assumed that the main antibiotic mechanism of action of organosulfur compounds is, together with its high permeability through phospholipid membranes, their interaction with certain thiol-containing enzymes in the microorganism. This reaction causes the inhibition of thiol-dependent enzymatic systems that could explain its activity against a wide range of Gram-negative and Gram-positive bacteria ,39. ConsAllium sativum) that exhibit antibacterial activity are oil-soluble organosulfur compounds, including allicin and allyl sulfides, among others [The reason for which PTSO showed higher antibacterial potential than other similar compounds could be related to its stability. The major phytochemicals derived from garlic  increased the relative percentage of survival of infected fish up to 51.6% [P. damselae [Pseudomonas fluorescens, presented lower mortality rates in groups that received 1 and 3% garlic extract in their diet [In the challenge trial with to 51.6% . Likewisdamselae . Other seir diet . This ineir diet ,51,52.Alliaceae-derived organosulfur compounds in different fish species and their relationship to mucosal immunity are well described. In summary, organosulfur compounds can induce immune responses and anti-inflammatory counterattacks and enhance the induced immunity by activating the TRPA1 and TRPV1 channels of enteroendocrine cells [Vibrio and Pseudomonas in the foregut and hindgut, whilst increasing beneficial Lactobacillus [The intestinal mucosa is the main barrier against the entry of pathogens, not only in fish but also in other species. Its protective function is conditioned by several factors, such as immunity and the composition of the intestinal microbiota. ,54. The ne cells ,55. Thisne cells ,56. Morebacillus . These mbacillus ,26,27.S. chrysophrii. The in vitro results showed a dose-dependent antiparasitic effect of both compounds, suggesting that PTS has the highest antiparasitic potential. Nonetheless, our results are similar to those reported in other in vitro and in vivo studies, in which the potential of Allium extracts against other monogenean gill parasites was analyzed. The in vitro exposure to garlic aqueous extract of the monogenean Gyrodactylus turnbulli caused the cessation of parasite movement, showing a positive correlation between garlic concentration and time to detachment and death. Moreover, guppy fish (Poecilia reticulata) fed with garlic-supplemented diets showed reduced mean prevalence and mean intensity of parasites [In our study, we also evaluated for the first time, both in vitro and in vivo, the effects of PTS and PTSO against arasites .Allium compounds, z-ajoene was the molecule that presented the highest antiparasitic activity and hypothesized that it could be due to the capability of this molecule to modify membrane proteins and lipid trafficking and enhance the expression of Th2 markers in the host cell that indicated a targeted immune response against parasites [Allium compounds against fish parasites such as Gyrodactylus turnbulli was reported to produce similar results to those obtained when using the antiparasitic agent levamisole [Allium compounds were tested against Spironucleus vortens, showed that dithiins presented the lowest MIC value against this parasite, followed by ajoene and allicin [S. chrysophrii when combined with other essential oils (EOs), such as carvacrol and thymol, and when included in the diet in a microencapsulated form, it reduces up to 78% of total infestation [S. chrysophrii [50 values, garlicin and eucalyptol showed intermediate effects, while r-camphor was the least effective. Nevertheless, another in vitro study demonstrated that curcumin was not effective against the parasite Neobenedenia girellae [Allium, to a greater or lesser extent, is effective for the control of gill parasites, although their degree of efficacy will depend, among other factors, on the species of the target parasite, the composition of the active ingredients and the dose of inclusion in the feed.Doan et al. reviewed the efficacy of different phytogenic compounds, including those derived from garlic, against different gill parasites, concluding that the observed antiparasitic capacity of these molecules could be due to the positive immunological effects of allicin in fish, resulting in improved fish immune responses against parasites . Similararasites ,60. Indevamisole . In addi allicin . Moreoveestation . These aysophrii . Among tgirellae . These sPTS and PTSO (95% purity) were supplied by DOMCA SAU  and dissolved in polysorbate 80 at 20%.The target strains used in this study were obtained from the Spanish Collection of Type Cultures (CECT) and the German Collection of Microorganisms and Cell Cultures (DSMZ) , and theS. chrysophrii parasites were obtained from donor fish provided by an offshore private fish farm, located in the Mediterranean Sea. Fish were placed in seawater containers with oxygen supplementation, and transported to the facilities of the Aquaculture Technology Centre of Andalusia, CTAQUA . Following this, 20 donor fish with an average weight of 120 g and a level of infestation of 1 parasite/fish in the first-gill arc were distributed in 2400 L truncated cone tanks provided with drains, with a layer of synthetic nylon fiber as a filter to retain the floating eggs released by the parasitized gilthead breams. This technique allowed us, after the hatching of eggs and in the presence of host fish, to reproduce the complete life cycle of the parasite.® antibiotic test discs, Buckinghamshire, UK) were impregnated with 20 µL of PTS or PTSO at a dose of 2.5, 5, 10, 25, and 50 mg/mL, and placed in the center of agar plates that were previously inoculated with the target strains. To achieve confluent growth after incubation, the bacterial suspensions used to inoculate the agar plates were adjusted to 1 × 106 CFU/mL. Three independent tests were carried out, in which each sample was tested in duplicate. The averages of all determinations were categorized as follows (disk diameter included): inhibition zones ≥ 20 mm were considered as strongly inhibitory, zones from 12 to 20 mm were considered as moderately inhibitory, and <12 mm were weak or not inhibitory [Two well-established procedures were carried out to assess the antibacterial activity of PTS and PTSO. The susceptibility of the target strains to both organosulfur compounds was screened by the disk-diffusion method proposed by Kirby–Bauer  and modihibitory ,70. The 5 CFU/mL. Then, 1:2 dilutions of both PTS and PTSO were prepared in 96-well microtiter plates to achieve the final concentrations, ranging from 4.88 to 10,000 μg/mL, and inoculated with 30 μL of the corresponding bacterial suspension. Each plate included a positive and negative control, without the tested compound or neither compound nor bacteria, respectively. Moreover, wells with a concentration of ceftazidime (8 µg/mL) were used as a second positive control. Absorbance was measured at 620 nm and cell growth was tested by culturing in agar plates. The lowest concentration of PTS/PTSO at which no growth was observed was defined as the MBC. MBC determination was performed in triplicate.The Minimum Bactericidal Concentration (MBC) of PTS and PTSO was determined using the standard broth microdilution method, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines . BrieflyS. chrysphriies larvae were obtained from fragments of nylon fiber inside the tanks of donor gilthead sea breams infected with the parasite. Fibers were continuously kept hydrated with salt water from the same tanks, until the evaluation of the antiparasitic activity. The larval stages of the parasite among the fibers were located by using a binocular loupe (20/40 magnification) and transferred to a 12-well culture plate , placing 10 larvae in each well. Parasites were exposed to PTS and PTSO at 0.5, 1 and 5 µg/mL and a control treatment with salt water was used. Additionally, a positive control with 4-hexylresorcinol at 1 µM was used to compare and detect dead parasites. All treatments were performed in triplicate. Viability was ensured at 0, 5, 30 and 60 min, considering the absence of mobility as a mortality indicator.S. aurata) were allocated in 400 L tanks with a recirculating RAS, equipped with physical and biological filters. The temperature was maintained at 21 ± 1 °C with a photoperiod regime of 12:12 h (light:dark) and daily water renewal of 10%. Water temperature , oxygen  and pH  were controlled daily. Salinity , ammonia  and nitrite  were monitored weekly. Fish were kept in these tanks until their use in antibacterial or antiparasitic in vivo challenge experiments. All animals were handled following the European Union Guidelines (Directive 2010/63/UE) for the use of laboratory animals.Juvenile gilthead sea breams (w/w (150 mg/kg). Once the meal was homogenized, the granulated fish feed was manufactured by SPAROS . The experimental diets followed a standard and commercially available basal formula for gilthead sea bream, containing fish meal, poultry by-product meal, oil seed, vitamin–mineral premix and soybean and rapeseed oil, among other ingredients . PTSO and PTS were added to the basal formula in a proportion 1:1; n = 90) were placed into 85 L tanks with the same initial biomass each  and randomly assigned to two experimental groups in triplicate . The control group was fed the non-supplemented diet during the whole experiment, while the ALLIUM group was fed a diet supplemented with 150 mg/kg of PTS/PTSO for 12 weeks prior to the start of the fish health challenge and afterwards, they were fed with the non-supplemented diet during the challenge. For infestation, the fish were previously anesthetized with 80 mg/L of tricaine methane sulfonate (MS-222) and were subjected to intraperitoneal injection with 100 µL of P. damselae subsp. piscicida DSM 22834 suspended in phosphate buffered saline (PBS), at a dose of 5 × 106 CFU/fish. The bacterial concentration in the inoculum was determined by ten-fold serial dilutions and further plating onto Columbia blood agar (Scharlau). The concentration of the bacterial suspension corresponds to the value of lethal dose 50 (LD50), which was previously determined. The control group of fish was inoculated with 100 µL of sterile PBS as a negative control.For this trial, juvenile gilthead seabreams . Fish were assigned to two dietary treatments (3 tanks per treatment), consisting of the non-supplemented diet for the control group and a diet that contained 150 mg/kg of a blend of PTSO and PTS in proportion 1:1; w/w for the ALLIUM group, over three weeks. Each week, 5 fish from each tank were euthanized with MS-222  at a dose of 200 mg/L and their gills were removed and placed on Petri dishes with salt water. Then, they were examined under a binocular loupe by combing the slides with the help of a needle, recording the number of parasites in each bronchial arch.To obtain a similar level of infestation, 150 healthy fish (average weight of 90 ± 0.1 g) were placed in tanks for 30 days with pieces of nylon fibers that contained parasite eggs. When the fish presented a homogeneous rate of infestation (around 10–15 parasites/fish), they were transferred to 6 tanks of 300 L at a ratio of 20 fish per tank  [Sparicotyle in the in vitro test and Photobacterium in the in vivo challenge were estimated by the Kaplan–Meier method and compared to controls by the log-rank test. The differences in the number of parasites per gill in Sparicotyle in the in vivo tests were compared using Student’s tests . The statistical significance level was accepted at p < 0.05.Data were statistically analyzed by using the Real Statistics Resource Pack software (Release 7.6) (ne 2022) . The effAlliaceae extracts against common pathogens in aquaculture was analyzed. Our results show that both phytogenics have antibacterial and antiparasitic activity. Although further studies including other stages and farm conditions should be performed, the increase in survival probability against P. damselae subsp. Piscicida and the reduction in the gill parasites S. chrysophrii of gilthead seabreams suggest the use of these allium-derived compounds as a natural diet strategy to prevent fish diseases in aquaculture.In this study, the capacity of the organosulfur compounds PTS and PTSO from


Malignant Catarrhal Fever is a globally distributed disease that is fatal to susceptible species such as cattle. Sheep represent the reservoir species, and the Mediterranean island of Sardinia, which hosts a large number of these animals, is one geographic area where virus can easily spread. The aim of our study was to investigate a case of Malignant Catarrhal Fever in a calf, also studying the prevalence of the virus responsible, Ovine Herpesvirus type 2, among sheep in Sardinia to further investigate the epidemiological aspects. The analyses performed were consistent among each other; indeed, the histological analysis revealed patterns of lesions, which are commonly reported in literature, in many tissue samples of the calf object of the study. We also found a considerable number of copies of viral genomes in all examined organs of the animal. Phylogenetic analyses suggested the possible occurrence of a unique genetic cluster that is widely distributed across the whole Italian territory. In conclusion, the present study provides a comprehensive overview on the Malignant Catarrhal Fever in an area where, despite the high prevalence of the Ovine Herpesvirus type 2 found among sheep, the sporadic occurrence of clinical disease in bovine should be still deeply investigated.Using a multidisciplinary approach, this report describes a clinical case of malignant catarrhal fever (MCF) occurring in a calf, which shared the pasture with sheep on a farm located in the island of Sardinia . We confirmed the conventional clinico-histopathological features of MCF, as well was the presence of Ovine herpesvirus type 2 (OvHV-2) DNA in several tissues, employing histological and virological investigations. The phylogenetic analysis revealed that this Sardinian OvHV-2 strain is genetically similar to all the other Italian strains. By Real Time PCR examinations of blood samples collected across Sardinia’s sheep population, which is considered the most important reservoir species, we discovered an OvHV-2 prevalence ranging from 20 to 30 percent. Despite the high prevalence of OvHV-2 in the Sardinian sheep population, clinical disease in bovine remains sporadic; further investigations are needed to understand the risk factors that regulate this epidemiological aspect.  Sheep-associated malignant catarrhal fever (MCF) is caused by ovine herpesvirus type 2 (OvHV-2), a virus belonging to the Macavirus genus of the Herpesviridae family . OvHV-2 Our report details a case of MCF occurring in a six-month-old Brown calf that happened on a farm in Northern Sardinia , where sheep also shared the pasture. Following the appearance of clinical symptoms, serum, EDTA samples, and nasal swab were collected for diagnostic investigations. Due to the severe clinical conditions, the calf was euthanized and submitted to necroscopy. The brain, thymus, lungs, small intestine, kidney, and liver were collected for histopathological, virological and bacteriological analysis. We were unable to detect the presence of OvHV-2 in the sheep sharing the pasture with the infected calf and for this reason, we aimed to outline the epidemiological situation of OvHV2 in Sardinia, where ovine farming is an important zootechnic activity with a total population of 3,000,000 sheep. The survey involved 1160 sheep reared in 24 different farms, spread across the island: 6 for the province of Sassari, 2 for Oristano, 7 for Nuoro, and 9 for Cagliari. EDTA blood samples were collected from healthy sheep with standard procedures.2O2 for 30 min at room temperature to quench endogenous peroxidase activity. The primary antibodies were incubated overnight at 4 °C. Immunohistochemistry (IHC) was performed using Dako EnVision kit  and counterstaining slides with Mayer’s haematoxylin. Each IHC run included adequate positive control tissues.For histological examinations, tissue samples were fixed in 10% neutral formalin. By using a routine protocol, paraffin embedded sections were obtained from each organ, cut into 4-µm-thick slices, and then stained with ST Infinity Hematoxylin & Eosin Staining System (Leica). The stained sections were examined using a light microscope with a magnification range of 50× to 400×. Furthermore, to define the local immune response related to MCF, CD immunophenotyping of inflammatory cells were carried out on selected kidney, liver, and brain tissue sections using primary antibodies CD3+ (DAKO AGILENT), CD79 (DAKO AGILENT) and PAX 5 (DAKO AGILENT), CD68 (BIORAD) and CD163 (BIORAD) specific for T lymphocytes, B lymphocytes, and macrophages, respectively. Sections were heat-induced in citrate buffer (pH 6.0)  to expose antigen epitopes, then slides were immersed in a solution of 0.3% HPasteurella spp. and Streptococcus spp. The ocular swab taken prior to the animal’s death was subjected to a culture test.Bacteriological examinations were performed by culturing on blood agar for TM Universal Probes Supermix  were used for this assay.Direct immunofluorescence techniques (IFA) using specific polyclonal sera (Kit IFA EUROKIT S.r.l.) were used to screen lung tissue for discarding Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BHV1), Bovine Herpes Virus 4 (BHV4), Bovine Respiratory Sincytyal Virus (BRSV), and Bovine Parainfluenza 3 (PI3) virus infections. The fluorescence was evaluated using an optical fluorescence microscope . Nucleic acid purification from thalamus, lung, liver, kidney, and intestine was performed using MagMax Core Nucleic Acid Purification Kit  in an automated sample preparation workstation MagMAX Express 96 (Applied Biosystems), according to the manufacturer’s instructions. Real Time PCR was used to detect the OvHV-2 genome in calf tissues and during the sheep screening whole blood samples, as described by D. Hussy et al. . Real Ti8 copie/µL). The cloned fragment was confirmed by Sanger sequencing on both strands by E. Sanger Sequencing 3500 series Genetic Analyzer (Applied Biosystem). The standard curve was created by placing the Cq values of eight serial 10-fold dilutions of p-OvHV-2 (1 × 107 to 1 × 101 copies/µL), performed in triplicate wells, against the log value of the number of copies of p-OvHV-2. The OvHV-2 copy number in each sample was determined using the Cq value to the standard curve and expressed as the number of copies target/µg total DNA extracted. 7900 Software SDS 2.4.1 (Applied Biosystems) generated The Cq value. The efficiency and R2 of the calibration curve were calculated. To generate the standard curve, we employed the plasmid pCR 2.1 TA Cloning (Thermofisher-Life Technologies) carrying a fragment of 131 bp of the ORF75 gene codifying FGAM-synthase of OvHV-2, named p-OvHV-2 . After the alignment, a manual check of the dataset was performed using Unipro UGENE v.35 [http://tree.bio.ed.ac.uk/software/figtree/ last access on 3 July 2022). A Principal coordinate analysis (PCoA) was also performed on a K2P [Phylogenetic analyses were performed on the ORF75 gene sequences. Therefore, a dataset (n = 72), which included the sequence isolated during the present study along with all the sequences of the ORF75 gene that are available on GenBank to date, was obtained (see sample codes in figure of the phylogenetic tree below reported for GenBank accession numbers and the geographic origin of the isolates). The sequences included were from Africa , Asia , the Americas , and Europe  and were mainly isolated in species belonging to the family of Bovidae, but also in a few individuals belonging to Suidae and Equidae. The sequences’ alignment was carried out using the Clustal Omega package   was 0.99 and the PCR-efficiency of 98%. With regards to the epidemiological investigation in Sardinia, we detected OvHV-2 DNA in all the flocks examined. We observed that 286 out of 1160 samples tested positive for OvHV-2 (24.6%), with a mean farm-level prevalence of 25%. More specifically, the prevalences were 20% in the province of Sassari, 21% in Cagliari, 23% in Oristano, and 33% in Nuoro.The tissues investigated for antigenic presence of BVD, BHV1, BHV4, BRSV, and PI3 resulted negative. We detected the OvHV-2 genome in all of the calf’s organs, including the brain, thymus, lungs, small intestine, kidney, and liver, using Real Time PCR, which was validated by Sanger nucleotide sequencing of amplicons. Results of the target genome copies number/1 µg of total extracted DNA obtained by qPCR in tested tissues organ give a viral load of 2.9 × 10Ovis canadensis) in USA (MN068217) and Canada (KX060582), respectively.The phylogenetic tree  evidenceWithin the main polytomic cluster, a further well-supported internal sub-cluster was present, with many sequences (70.27%) from North Africa and Asia , and only a few (29.73%) from Europe, South Africa, and South America . Apart from those included in the internal sub-cluster, the other sequences included in this large polytomic cluster, which also included the Sardinian strain isolated in the present study, were almost all from Italy, with only a few from the Americas, Asia, Africa, and Europe . The PCoA  explaineIn our report, histological and virological analysis, as well as phylogenetic inferences, are consistent among each other, thus confirming the presence of OvHV-2 in a calf with the distinctive clinical signs of the disease. To perform a proper histological diagnosis of MFC, representative and specific tissues must be collected during a calf necropsy. In this regard, the sampling of mesenteric lymph nodes, small intestine, lung, eye, carotid rete mirabile, and urinary bladder are typically suggested . MoreoveIt is broadly accepted that CD163+ macrophages, are considered anti-inflammatory phenotype. Regarding our case, we speculate that OvHV-2 could be capable of inducing anti-inflammatory phenotype in the macrophages of the host for helping the disease progression. Interestingly, a study on lung pathology in man during acute respiratory distress syndrome caused by SARS-CoV-2 revealed an accumulation of CD163+ macrophages which correlated positively with the severity of the disease by intensifying fibro-proliferative response .Pasteurella spp. and Streptococcus spp. can occur, although they seem to be not responsible for the observed symptoms. Although it was not possible to extend the investigation to all animals of the farm where the analyzed calf lived to trace the source of infection, in this study, we found 27 positives out of 98 samples examined (27.5%) in the same geographical area. Phylogenetic and genetic clustering analyses evidenced that the sequence of the gene ORF75 for OvHV-2 isolated in Sardinia is genetically similar to all of the other Italian strains present on GenBank. This finding suggests the possible occurrence of a genetic cluster that is widely distributed across the whole Italian territory. This condition is quite common in DNA viruses as OvHV-2 due to their low mutational rate [The evaluation of the number of genomic targets in the calf investigated organs allowed us to highlight that a considerable number of copies of viral genomes are present in the host brain, specifically in the thalamus, slightly less than in the lung, but comparable to those found in the liver, kidney, and intestine. As a result, the high number of target viral genome copies retrieved in our calf’s brain suggests that this organ could also be considered as an elective organ of choice for MCF diagnosis. Our bacteriological results showed that in the case of MCF infection, opportunistic infections from nal rate ; on the nal rate .In conclusion, this work provides a comprehensive clinical-pathological and virological characterization of a case of MCF in a calf, as well as one preliminary estimate of OvHV-2 prevalence in sheep across the whole Mediterranean island of Sardinia. Given the epidemiological findings on the high circulation of the virus in the territory, which are in contrast to the sporadic clinical detection of the disease in bovine, we believe that further investigation is needed to help understand the risk factors that governing this aspect. Indeed, it is likely that the disease is underestimated and should be included in the differential diagnosis of bovine diseases.


Codes included type of study, methods, use of theory, and whether and how the relationship between social media and alcohol use was tested, among others. In addition to providing descriptive findings, the current study compared the findings across studies that primarily focused on advertising and marketing, self-generated user-generated content (UGC), other-generated UGC, social media uses and affordances, and a mixture of more than one type of content/focus. Most articles used quantitative methods (77.94%), which is followed by qualitative methods (15.20%), mixed methods (6.37%), and 0.49% that did not fit in any of the methods categories. Of the studies that tested the relationship between social media use and alcohol consumption, an overwhelming majority found that relationship to be positive (93.10%). The results of the present study provide a comprehensive understanding of past findings regarding social media and alcohol consumption and provide important future research suggestions.Many studies have looked at the relationship between social media and alcohol consumption. There is a need for a comprehensive review that synthesizes the results of past research to systematically understand the relationship between social media use and alcohol consumption. The present systematic literature review synthesizes the findings from global social media and alcohol use studies ( The rise of social media use, specifically among adolescents and young adults, has not only revolutionized human communication but has also magnified social and peer influences regarding social, political, and health-related issues . One impThe relationship between social media use and alcohol use has been examined from multiple disciplinary perspectives, including media and mass communication, psychology, health, and socio-technical system approaches, leveraging a diverse set of methodologies that cover behavioral and attitudinal research (self-report), psychophysiological and eye-tracking, as well as analysis of large-scale data sets using information system and computer science methodologies . The relationship between the two behaviors can be explained using social learning theory and social cognitive theory, both of which emphasize that humans learn or change their behaviors by observing others ,6. SeeinExposure to alcohol-related content is not the only affordance on social media platforms . On sociThe theoretical understanding of the relationship between alcohol use and social media use, outlined above, provides avenues for practical and health-related applications, risks, and concerns. Over a 10-year period (between 2012 and 2022), the average time spent on social media daily grew by over 63% to 147 min  . At the To date, there are two systematic literature reviews (SLR) ,15 and oAs noted above, to date, there are two SLRs ,15 and oThe two SLRs relevant to this topic of inquiry ,15 coverCurtis and colleagues  conducteThus, in sum, previous reviews and meta-analyses have significant implications and have made relevant contributions. However, these reviews are also either broadly focused on overall risky behaviors including alcohol use, narrowly focused on only exposure to commercial alcohol content, limited in methodological scope, or restricted in the extent of sources. This current SLR addresses all these gaps by including and comparing both UGC and commercial alcohol-related content on social media as well as studies focusing on linking general aspects of social media use to alcohol consumption. Additionally, the SLR approach followed in the current study expands the inclusion criteria to non-quantitative research approaches as well as studies published in a variety of publication venues. We also examine this phenomenon across different age groups, thus providing a more comprehensive overview of the existing literature. By analyzing not only the results of these studies but also their methodological and theoretical approaches, this SLR can provide directions for future research. Lastly, qualitative work, theoretically informed and designed studies, and peer influence are included in this review, because as Groth et al.  suggesteIn the current study, we aim to compare studies related to alcohol advertising and marketing, self-generated user-generated content (UGC), other-generated UGC, and social media use and affordances. Therefore, it is essential that we provide conceptual definitions of the major concepts relevant to this review. Social media refers to “Internet-based channels that allow users to opportunistically interact and selectively self-present, either in real-time or asynchronously, with both broad and narrow audiences who derive value from user-generated content and the perception of interaction with others”  (p. 50).Advertising and marketing content refers to paid, owned, or earned  content distributed through social media with the intent to persuade someone to adopt or change their attitudes and/or behaviors . As it rOur study also identified two types of UGC: self-generated and other-generated. Krumm, Davies, and Narayanaswami  define UThe last type of studies deals with studies that examined general social media use or entailed aspects of the platform’s affordances. This type includes studies that measured the amount of time spent on social media, the size of one’s network of friends on the platform, and specific uses of social media .Aim (1) To describe the prevalence, methods, and theories related to alcohol-related social media studies. Aim (2) To describe the findings regarding the relationship between social media and alcohol use. Aim (3) To compare studies focusing on user-generated and commercial alcohol content on social media. These aims are translated into three overarching research questions:RQ1: Prevalence, methods, and theories:RQ1a: Prevalence: What is the prevalence of alcohol-related social media studies over time, by location, and by funding source?RQ1b: Methodological approaches: What are the methodological approaches  employed in alcohol-related social media studies?RQ1c: Theoretical approaches: What are the theoretical approaches employed in alcohol-related social media studies?RQ2: Test of the relationship between social media and alcohol use:RQ2a: Nature of relationship: What was the nature of the tested relationship between social media and alcohol use ?RQ2b: Findings: What findings did studies report regarding this relationship?RQ3: Comparing UGC and Commercial studies:How do alcohol UGC and commercial content compare in terms of (a) prevalence, (b) methods, (c) sample characteristics, (d) measures, (e) theoretical approaches, and (f) their test of the relationship between social media and alcohol?This SLR has three overarching goals: Per Fink , . Trained researchers examined each database list of articles and performed initial screening of the records to ensure that the studies retrieved focus on both alcohol use and social media use. Upon initial screening and assessment of the relevance, we identified a total of 1119 studies as a combined sample across the different databases. The removal of duplicates yielded a sample of 467 studies (see We adopted the PRISMA approach for conducting SLR . We useddies see . After tn = 291), a team of four independent coders pretested the coding scheme with five articles. The four coders met and resolved differences, which has yielded a revised version of the codebook. The four coders then independently coded 10% of the sample, which resulted in an 89% average agreement among the coders. During this coding phase, coding categories with percentage agreement lower than 75% we assessed closely, and the coders refined their interpretation of these categories to ensure reliable coding of the entire sample. The articles coded for inter-rater reliability were then integrated into the larger data set by recording each category by majority rule. In a minority of cases, where agreement was split among the coders, one of the coders re-examined the article and recoded that category based on the refined understanding of the codebook.Upon thoroughly screening articles  alcohol consumption measures, social media platform, and social media content. For a full description of the codebook, refer to n = 108; 52.94%) included a mixture of advertising/marketing content, self- and other-generated UGC, and social media use and affordances measures, which were followed by 51 studies (25.00%) that focused solely on self-generated UGC, 22 studies (10.78%) that focused on other-generated UGC, 14 studies (6.86%) that focused on social media use and affordances, and nine studies (4.12%) that focused exclusively on alcohol advertising and marketing (see 2(4) = 164.28, p < 0.001.Of the 206 studies, two were excluded as they did not fit any of the content type categories; thus, 204 studies were retained for all subsequent analyses. Most studies  =n = 167, 81.86%), which were followed by research abstracts , conference papers , graduate theses and dissertations , and five book chapters (2.45%), respectively, χ2(4) = 489.33, p < 0.001  reported findings from a single study, with only four articles that reported findings from two studies (1.96%); the remaining two studies comprised a conceptual piece published in the Youth Drinking Cultures in a Digital World book [2(2) = 372.82, p < 0.001.Most coded articles were classified as research articles published in peer-reviewed journals  were situated in the United States, with the remaining 87 studies distributed across the following countries, respectively: Australia , the United Kingdom , New Zealand , Belgium , Norway , the Netherlands , Mexico , two studies each (0.98%) from Canada, Sweden, and Switzerland, and a single study each (0.49%) from Italy, Korea, and Thailand. Additionally, 12 studies (5.88%) focused on multiple countries, with five of those focusing on Australia and India, one study conducted in Belgium and the United States, one study in England and Australia, one study in South Korea and the United States, one study in the United Kingdom, Ireland, and Australia, one study from the Netherlands and Germany, and two global studies with multiple countries. A total of 12 studies (5.88%) did not specify a geographic location for the conduct of the study. Over half of the studies  reviewed disclosed support from funding entities, including governmental agencies, foundations, and other sources.Over half of the studies  used quantitative methods, followed by 31 articles that used qualitative methods (15.20%), 13 articles that used mixed methods (6.37%), and 1 article (0.49%) that did not follow a particular research methodology, χ2(3) = 313.88, p < 0.001. For the studies that used quantitative methods (including those leveraging mixed-method approaches), 79 (48.77%) used survey methodology, followed by content analysis , big-data mining , experimental research , and multiple quantitative methods , χ2(4) = 92.51, p < 0.001. Of the 35 studies that used qualitative methods, 14 used focus groups (40.00%), four used interviews (11.43%), five used textual analysis (14.29%), eight used multiple qualitative methods (22.86%), and four articles that used qualitative methods not listed in our coding scheme (11.43%), χ2(4) = 10.29, p = 0.04. Of the articles leveraging quantitative methodologies and mixed methodology, 71 used cross-sectional designs, while only 21 used longitudinal research designs, χ2(1) = 27.17, p < 0.001, thus indicating a much higher prevalence of cross-sectional than longitudinal designs  were conducted online, followed by non-human subjects , field studies , and studies conducted in a lab or research facility . An additional 17 studies (8.33%) did not specify the context of the research, χ2(4) = 100.51, p < 0.001  recruited participants below the age of 21, 61 studies (29.90%) recruited adult samples (over 21 years old), and 16 studies (7.84%) recruited samples of underage youth and adults. The remaining 82 studies (40.20%) did not indicate the age of participants, bearing in mind that some of these studies are non-human subjects studies, χ2(3) = 45.53, p < 0.001  included a reference in the method section related to the sample’s gender distribution, χ2(1) = 4.41, p = 0.04.In terms of the age ranges of participants, we classified studies based on the minimum legal age of 21 to purchase of alcoholic beverages in the United States. Based on this, over one-fifth of the sample  exclusively recruited drinkers, χ2(1) = 116.26, p < 0.001. Less than half of the studies  relied on self-report measures of alcohol use, and 106 studies (51.96%) did not explicitly indicate the way alcohol use was measured, with only one study using unobtrusive measures of alcohol use such as blood alcohol concentration (BAC) and breathalyzer, χ2(2) = 99.62, p < 0.001. Of the 204 studies, 83 (40.69%) measured alcohol use within a specific timeframe, χ2(1) = 7.08, p < 0.01. Thirty-one studies (15.20%) specifically used the alcohol use disorders identification test (AUDIT) to index alcohol use, with 47 additional studies (23.04%) that relied on alternative clinical and standardized measures of alcohol such as the Daily Drinking Questionnaire (DDQ), NIDA Modified ASSIST substance use screener, Quantity Frequency Index, and the timeline follow back (TFLB) method. Roughly four in 10 studies  measured perceptions of alcohol use among peers and friends were measured, χ2(1) = 12.26, p < 0.001. About one-fifth of the studies reviewed  included a social norms measure related to alcohol use, χ2(1) = 75.27, p < 0.001. Seventy-six studies (37.25%) included a reference of excessive drinking, χ2(1) = 13.26, p < 0.001. An extreme majority of studies did not reference short- and long-term risks associated with drinking , χ2(1) = 169.59, p < 0.001, where nine studies included references to negative consequences of alcohol use, which included a single study focused on long-term consequences, seven focused on short-term consequences, and a single study that focused on both long- and short-term consequences. A minority of the studies reviewed  measured the use of other drugs concurrently with alcohol use, χ2(1) = 104.49, p < 0.001. Finally, only four studies focused on celebration drinking, χ2(1) = 188.31, p < 0.001.Social Media Measures. The top social media platforms in terms of use [n = 38, 18.63%) referenced general social media use in the studies, and a similar portion of the sample  included multiple social media platforms for investigation, and 10 studies (4.90%) investigated other platforms such as Myspace and Reddit, χ2(6) = 123.08, p < 0.001 (see 2(1) = 93.35, p < 0.001. Thirty-seven (18.14%) of the studies included a measure of time spent on social media, χ2(1) = 82.84, p < 0.001. Additionally, 20 studies (9.80%) measured the number of social media friends, χ2(1) = 131.84, p < 0.001.s of use  were als.001 see . Of the n = 139, 67.16%) of the coded articles did not include any theoretical framework. The 67 studies (32.84%) using theories leveraged 39 different theories, theoretical models, frameworks, and concepts, χ2(1) = 24.02, p < 0.001 (see Over two-thirds (As noted in 2(2) = 121.94, p < 0.001. On the other hand, of the 111 articles that measured social media use, 89 articles (80.18%) measured it as a predictor or IV, 16 articles (14.41%) measured it as criterion/DV, and six articles (5.41%) measured it as a different type of variable, χ2(2) = 110.97, p < 0.001. Thus, most studies measured social media use as a predictor/IV and alcohol use as a criterion/DV. Exceptions included studies where the relationship was reciprocal, alcohol use was a control variable, or either construct regarded as a mediator or moderator.To test RQ2a we coded alcohol use and social media use in each study where the relationship was formally tested as predictor/IV, criterion/DV, moderator, mediator, or control variable. For convenience’s sake, a three-category variable was created for each coded variable where 1 = predictor/IV, 2 = criterion/DV, and 3 = Other, which included cases where either variable was a mediator, moderator, or control variable. Additionally, we added instances where a correlational (often bivariate) relationship was tested; thus, each variable was regarded as both predictor and criterion. Of 96 studies that measured alcohol use, an overwhelming majority of 83 articles (86.46%) measured alcohol use as a criterion or dependent variable, only six (6.25%) articles included it as a predictor or independent variable, and seven others (7.29%) included it either as a mediator, moderator, control, or both predictor and criterion, χn = 81, 93.10%), five studies (5.74%) did not find a relationship between the two variables, and a single study (1.15%) found the relationship to be negative in direction, χ2(2) = 140.14, p < 0.001.A total of 163 examined the relationship between social media and alcohol use (only quantitative studies and mixed methods with quantitative approaches). With that in mind, of the 162 cases viable for coding here, 87 studies (53.70%) formally tested the relationship between the two constructs, and the remaining 75 studies did not conduct a formal test of that relationship. Of the 87 studies that formally tested the relationship, an overwhelming majority found that relationship to be positive  examined in the study. Descriptive statistics (frequencies and percentages) along with FET test value and Studies did not vary significantly in terms of publication venue, with the majority of studies across social media content and use types appearing in peer-reviewed journals. Regarding the country where the study was conducted (sample selection), all SMCU types studied, except mixed content types’ studies, were more heavily focused on the United States, where the mixed content types’ studies had a greater prevalence of non-U.S. samples. Studies across SMCU types were somewhat evenly split in terms of funding.Study types were uniformly distributed in terms of the research methodology employed in the study (no significant differences), with most studies using quantitative methods. However, for studies employing quantitative methodology, the differences were significant in that advertising/marketing studies were most experimental and content-analytic; self-UGC studies were mostly survey-based, followed by content analytic, big-data and multiple-method studies ; other-UGC studies were mostly survey-based, followed by experimental, content analytic, big data, and multiple methods; SM Use and Affordances studies were mostly survey-based with one study using multiple quantitative methods; about four in 10 of the SMCU types studies were survey-based, followed by a quarter that used content analysis, one in five using big data, and less than 10% using each experimental and multiple methods. No significant differences were detected for qualitative study categories, research design , and distribution method (mostly online).Differences between SMCU types in terms of participant age were marginally significant. Although a minority of studies across SMCU types focused on participants younger than 21 years old, the different types slightly varied: one advertising/marketing study, one-third of self-UGC studies, over one in five of other-UGC studies, and less than one in five of mixed SMCU types studies focused on individuals under the age of 21. SMCU types also varied in terms of reporting the gender distribution of participants, where the majority of studies in all types, except mixed-SMCU types, reported the gender distribution of their samples; over half of mixed SMCU types studies did not report the gender distribution of the study sample. Finally, the SMCU types did not differ in terms of focusing solely on alcohol users (drinkers), where most studies recruited both drinkers and non-drinkers.Studies across the different SMCU types mostly relied on self-report measures of alcohol use. Studies varied in terms of measuring alcohol in a specified timeframe , where advertising/marketing, other-UGC, and mixed SMCU types studies had a higher frequency of studies that did not include a time-based measure of alcohol use (between two-thirds and seven in 10), while most studies in the self-UGC and SM use and affordances categories included a time-based measure of alcohol use. With regard to use of clinical alcohol use measures , self-UGC studies had the highest prevalence of studies that used AUDIT and other clinical measures , where most other studies did not specify a clinical measure of alcohol use. Advertising/marketing studies and mixed SMCU types studies did not include a measure of drinking among reference groups and/or friends, whereas, about half the studies coded as self-UGC, other-UGC, and SM use and affordances included such measures. Most studies in all SMCU types categories (over 85%), except SM use and affordances, did not integrate measures of other drugs in the study, where over four in 10 SM use and affordances studies included such measures. SMCU types did not vary in terms of focus on social norms, excessive drinking, negative consequences of drinking, and celebration drinking.Regarding social media use measures, the different SMCU types varied significantly in terms of the social media focus in the study and inclusion of social media use frequency (time) but not in terms of including a measure of the number of social media friends. For the social media context of the study, Facebook was the most frequent content for advertising/marketing and self-UGC studies, while other SMCU types were more distributed across the different platforms. In contrast to other SMCU types that did not include a measure of social media use frequency (time), over half of SM use and affordances studies included a social media use time measure.All SMCU types, except other-UGC and SM uses and affordances, had a higher frequency of studies without a theoretical framework. Over half of other-UGC and half of SM uses and affordances studies included a theory.We observed uniformity across SMCU types in the designation of alcohol use as a criterion or DV and social media use as a predictor or IV. SMCU types varied significantly regarding the formal testing of the relationship between social media use and alcohol consumption: nearly six in 10 advertising/marketing and mixed SMCU types studies did not test the relationship, while most self-UGC (nearly three-quarters), other-UGC , and SM uses and affordances (over eight in 10) tested it. When that relationship was tested in the studies across SMCU types, it was found to be positive in nature.This SLR builds upon and extends three previous reviews of the literature on social media use and alcohol ,15,16,17Regarding the first aim , where alcohol use was not measured within a timeframe. Additionally, most studies did not leverage clinical and established measures of alcohol use , did not measure perceptions of drinking among different reference groups, including friends, and shied away from leveraging the social norms approach as an operational framework for social influence. The lack of uniformity in operationalizing alcohol use needs further attention by researchers in this field, especially that clinically acceptable measures could be necessary to further situate this this area of inquiry within the public health domain. Secondly, the lack of social contextualization of alcohol consumption, and especially given the ‘social’ nature of social media, is a missed opportunity for extending our understanding of this phenomenon beyond the individual focus by incorporating social and interpersonal relationships as factors driving both alcohol and social media uses. Similar concerns emerge where much of the research reviewed here disregards measures of excessive drinking, negative consequences and risks associated with drinking, concurrent alcohol use with other drugs, and celebration drinking. Such gaps in the literature should inform the future research agenda to expand the focus from the individual general use of alcohol to more socially informed aspects of alcohol consumption and overuse, along with other risky and regulated substances.Our findings showed that Facebook maintained the lead in terms of the context of examining the phenomenon at hand, with platforms such as Twitter, Instagram, YouTube trailing behind. It is worth nothing that a considerable portion of the studies examined social media use as a general area of use without specifying any platform as the context of the study as well as a portion of the studies that examined multiple platforms concurrently. It is worth mentioning that newer social media platforms, such as TikTok, have not been represented in the sample of studies reviewed here and there was a low prevalence of studies on exponentially growing platforms such as Instagram and Snapchat, which was plausibly due to the recent increased popularity of such platforms, thus not leaving much time for the academic literature to catch up with these trends. One noteworthy finding is the low prevalence of studies measuring time spent on social media and the number of friends on social media as metrics to index social media use. It is apparent that social media use has been dealt with in a categorical way, outside the boundaries of more robust measures of extent of use and other important factors that index this use .One of our major findings relates to the severe lack of theory-based research in this domain. Less than one-third of the studies leveraged a theoretical framework, and these theories were dispersed across different disciplines. The profile of studies included in this SLR spans multiple disciplines, and in several cases, the research is published in interdisciplinary journals, yet much of the theories used tend to examine the phenomenon on the psychological level. This could be a plausible explanation for the lack of clear theoretical foundations of the research in this area. However, as this field matures, it is critical for more theory-based investigations that not only serve to predict aspects of human and technology-related behaviors but rather could aid with designing effective strategies and interventions to combat alcohol-related risks that are further elevated within the social and digital media environments. Such practical extensions of this body of literature could also be informed by greater emphasis on sociological and critical theoretical frameworks to examine the impact of social and cultural structures, values, and practices on the relationship between alcohol use and social media use.To address the second aim in our SLR, we examined how the relationship between social media use and alcohol consumption was operationalized and tested. One of the most intriguing findings in our SLR deals with providing a clear determination that social media use is an antecedent to alcohol consumption within this body of literature. Despite the low frequency of experimental research in our sample, replicative evaluation of the associative relationship between the two constructs equips our team to propose a causal order for this relationship, albeit mostly reliant on associative examination of that relationship: social media use predicts alcohol consumption, and this relationship is squarely positive, in that greater social media use is associated with higher alcohol consumption.Finally, to address this study’s third aim, we compared the corpus of studies as a function of the social media content and use (SMCU) types see . We examIn terms of divergence, there are several important findings to discuss. First, the divergence in terms of the types of quantitative methods used in this domain is important to drive cross-pollination across disciplines. We see that advertising and marketing research in this area tends to be heavily focused on experimental work, yet other types favor survey-based research. It is important to reflect methodological diversity within both quantitative and qualitative approaches to better formulate a comprehensive understanding of this socio-technical/health phenomenon. Second, the divergence also points to a lack of cross-disciplinary standardization of how the two constructs were operationalized. Wider discussions within the field and across fields is necessary to further harmonize these methods of operationalization. Finally, our review points to methodological divisions in terms of examining the two constructs within the same study. Much of the research did not formally test the relationship, despite alluding to it. Whether conducting a quantitative or qualitative study, it is important for research in this domain to further showcase the extent to which the two constructs are related. This is also important regarding pushing the boundaries in what we termed here as big-data analyses, where researchers, with considerable mastery, mined and analyzed (through computer- and human-based coding) large data sets of social media posts related to alcohol. A few of the studies attempted to link social media content to community-related alcohol use and incidents of alcohol overuse . More of the latter type of research is needed within this domain, where the emphasis should evolve into highlighting the impacts of digital touchpoints on individuals and societies.To address this topic, we provide an overview of topics previous reviews called for in new research on social media and alcohol, and then within them, we offer insights garnered from the results of this systematic review.First, previous reviews on social media and alcohol use have called for longitudinal studies that can establish the causal order of the variables ,15,16, aSecond, previous reviews called for greater investment in theory-driven research in this area of research, the lack of which was evident in our review, thus intensifying the need for more theory-based research here. It is worth mentioning that our review highlighted a strong diversity of theoretical frameworks across academic disciplines that examined the relationship between social media, which we deem as a point of strength for this area of research. However, we argue that there is a need to, potentially, organize the theorizing in this area of research across epistemological approaches and greater distribution among different disciplines, especially that most theories used in the literatures were psychological in nature. Additionally, Groth et al.  noted thThird, similar to Groth et al. , our finFourth, Facebook still maintains the lead in terms of study context. While Facebook is still the leading social media platform use worldwide, recent increases in popularity, platform substitution, and prevalence of newer uses of social media platforms have emerged, specifically, among younger users. With that in mind, future research should reflect those dynamic changes in the social media scene and account for the changing rituals and habitual uses of these platforms. For example, Snapchat, which entailed a low prevalence of studies in our sample, has been increasingly used for sharing alcohol-related content and messages, specifically due to its affordances related to disappearing content after a short period of time, thus alleviating privacy concerns with sharing such type of risky content. Another emerging platform worth examining is TikTok, which has exponentially increased in popularity over the past few years. It is also worth mentioning that for certain types of studies, specifically content-analytic and big data studies, a higher prevalence was observed for platforms such as Twitter, which was plausible due to the ease of extracting data from such platforms in comparison to other platforms with high API restrictions . Finally, it is important to expand on how researchers in this field measure social media use to include more robust measures of time spent on social media and number of friends, including server-based accounts of social media use. Future research should also reflect the growth and evolution in terms of affordances and functions of different platforms to further decipher the most important pain points related to the impact of social media use on alcohol consumption.Fifth, despite our efforts in the current SLR to include a wide range of databases, most of the studies were in English and centered in the United States and other Western countries. Alcohol use, overuse, and abuse is a global public health concern, which causes three million deaths annually, representing 5.3% of all deaths globally . AcademiIn summary, our coding of the literature by the types of content studies provided us with several intriguing operational and theoretical insights into the large body of research focusing on the use of social media and how that relates to alcohol use and overuse. Throughout our coding, we extrapolated differences between advertising and marketing research in how it reflected the relationship between alcohol advertising and marketing, thus shedding the light on persuasive branded communication. The two coding categories related to UGC stratified the focus into self-generated social media posts and other-generated posts, thus highlighting the interpersonal  nature