Members Of Genus Research Articles

The description of a novel Gloeothece species (Chroococcales, Cyanobacteria) from China, based on a polyphasic approach

Cyanobacteria, with strong adaptability, occupied an important position on the earth all the time. In this study, the strain of coccoid cyanobacteria (CHAB 5847) was isolated and purified from Guizhou Province, China. The phylogenetic and taxonomic characteristics of CHAB 5847 were studied using polyphasic approach combining morphological, ecological and molecular data. Based on phylogenetic analysis of 16S rRNA gene sequence, the studied strain occupied a unique position, and clustered in Gloeothece clade. The 16S rRNA gene sequences of two clones of the studied strain shared 93.1%–98.1% similarity to other Gloeothece species, which supports well it to be a new member of Gloeothece genus. Furthermore, the unique pattern of D1-D1′, Box-B and V3 helix of the 16S–23S rRNA internal transcribed spacer (ITS) secondary structure revealed that the strain represented a novel species. These results support the establishment of a new species of Gloeothece genus with the name G. guizhouensis sp. nov.

Acute nitrite exposure causes gut microbiota dysbacteriosis and proliferation of pathogenic Photobacterium in shrimp

Nitrite exposure has become a significant concern in the aquaculture industry, posing a severe threat to aquatic animals such as shrimp. While studies have reported the adverse effects of nitrite on shrimp growth, the part played by the gut microbiota in shrimp mortality resulting from nitrite exposure is poorly understood. Here, the effects of nitrite on shrimp gut bacterial community were investigated using 16S rRNA amplicon sequencing, bacterial isolation, genomic analysis, and infection experiments. Compared to the control_healthy group, changes in the bacterial composition of the nitrite_dead group were associated with reduced abundance of specific beneficial bacteria and increased abundance of certain pathogenic bacteria. Notably, members of the Photobacterium genus were found to be significantly enriched in the nitrite_dead group. Genomic analysis of a representative Photobacterium strain (LvS-8n3) revealed a variety of genes encoding bacterial toxins, including hemolysin, adhesin, and phospholipase. Furthermore, it was also found that LvS-8n3 exhibits strong pathogenicity, probably due to its high production of pathogenic factors and the ability to utilize nitrite for proliferation. Therefore, the proliferation of pathogenic Photobacterium species appears pivotal for driving shrimp mortality caused by nitrite exposure. These findings provide novel insights into the disease mechanism in shrimp under conditions of environmental change.

Senecavirus cetus a novel picornavirus isolated from cetaceans represents a major host switching to the marine environment

Senecavirus A (SVA), an emerging virus that causes vesicular disease in swine, was, until recently, the only member of the Senecavirus genus (Picornaviridae). Here, we report the isolation and complete genome sequence of two isolates of cetacean picornavirus 1 (Senecavirus cetus), a novel picornavirus species of the Senecavirus genus from dead stranded cetaceans from Alaska. One isolate was from a harbor porpoise stranded in 2017, and another from a beluga whale, stranded in 2019. Whole-genome sequencing of Senecavirus cetus strains showed a genome-wide nucleotide identity of 98.8% and a genome size of 7455 nucleotides. The Senecavirus cetus genomes are most similar to SVA with a 58.3% genome-wide pairwise nucleotide identity. Infection of eleven available cell lines from terrestrial and aquatic animals showed that beluga and sheep cells were susceptible to infection by Senecavirus cetus. Phylogenetic and ancestral state reconstruction analyses supported the novel virus being a member of the Senecavirus genus and provided the first evidence of Senecavirus-like picornavirus infecting marine mammals and likely descending from a terrestrial host ancestor. These discoveries provided important information on the evolutionary relationships and taxonomy of picornaviruses and increased our understanding of the genomic characteristics and potential host range of Senecavirus cetus.

Prevalence, genetic characteristics and economic losses of Foot-and-Mouth Disease Virus (FMD) in global and Bangladesh context: A Review

Foot and Mouth disease virus (FMD) is a transboundary animal disease (TAD) affecting global cloven-hooved wild and domesticated animals. It is recognized as a significant economic infectious disease in the livestock business. Domesticated animals such as goats, sheep, cattle, pigs, and buffalo are vulnerable hosts for FMD. This RNA virus is classified as a member of the Aphthovirus genus in the Picornaviridae family. It encompasses around 60 subtypes and is divided into seven serotypes: C, O, A, SAT1, SAT2, SAT3, and Asia1. There is no cross-protection across different serotypes, and occasionally, the effectiveness of vaccination may be restricted, even when targeting the same serotype. Therefore, it is crucial to identify and classify the viruses currently in circulation, mainly when vaccination is employed for disease management. Hence, it is necessary to administer multivalent vaccines with genetically matching vaccines in circulation to ensure adequate protection through immunization. Bangladesh has a high cattle population, and FMD leads to significant economic losses for farmers. Serotypes A, O, and Asia 1 are circulating in Bangladesh. The most common serotype is O, although Bangladesh has reported multiple serotype infections. Strategic vaccination, immunity screening, and outbreak surveillance are being examined for FMD management in Bangladesh. This review analyses FMD epidemiology, genetics, diagnostics, economic impacts, and prevention-control efforts worldwide, emphasizing Bangladesh. Finally, the study recommends FMD management advice for endemic settings.

Enhancing the understanding of coinfection outcomes: Impact of natural atypical porcine pestivirus infection on porcine reproductive and respiratory syndrome in pigs

Atypical porcine pestivirus (APPV) is a novel member of the Pestivirus genus detected in association with congenital tremor (CT) type A-II outbreaks and from apparently healthy pigs, both as singular infection and as part of multi-pathogen infections. 'Classical' pestiviruses are known to cause immunosuppression of their host, which can increase susceptibility to secondary infections, severely impacting health, welfare, and production. To investigate APPV's effect on the host's immune system and characterise disease outcomes, 12 piglets from a natural APPV CT type A-II outbreak were experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV), a significant porcine pathogen. Rectal temperatures indicating febrile responses, viremia and viral-specific humoral and cellular responses were assessed throughout the study. Pathological assessment of the lungs and APPV-PRRSV co-localisation within the lungs was performed at necropsy. Viral co-localisation and pathological assessment of the lungs (Immunohistochemistry, BaseScope in situ hybridisation) were performed post-mortem. APPV status did not impact virological or immunological differences in PRRSV-infected groups. However, significantly higher rectal temperatures were observed in the APPV+ve/PRRSV+ve group over four days, indicating APPV increased the febrile response. Significant differences in the lung consolidation of the apical and intermediate lobes were also present, suggesting that APPV co-infection may augment lung pathology.

Computational discovery of tripeptide inhibitors targeting monkeypox virus A42R profilin-like protein

Monkeypox is an infectious disease caused by the monkeypox virus (MPXV), a member of the Orthopoxvirus genus closely related to smallpox. The structure of the A42R profilin-like protein is the first and only available structure among MPXV proteins. Biochemical studies of A42R were conducted in the 1990s and later work also analyzed the protein's function in viral replication in cells. This study aims to screen tripeptides for their potential inhibition of the A42R profilin-like protein using computational methods, with implications for MPXV therapy. A total of 8000 tripeptides underwent molecular docking simulations, resulting in the identification of 20 compounds exhibiting strong binding affinity to A42R. To validate the docking results, molecular dynamics simulations and free energy perturbation calculations were performed. These analyses revealed two tripeptides with sequences TRP-THR-TRP and TRP-TRP-TRP, which displayed robust binding affinity to A42R. Markedly, electrostatic interactions predominated over van der Waals interactions in the binding process between tripeptides and A42R. Three A42R residues, namely Glu9, Ser12, and Arg38, appear to be pivotal in mediating the interaction between A42R and the tripeptide ligands. Notably, tripeptides containing two or three tryptophan residues demonstrate a pronounced binding affinity, with the tripeptide comprising three tryptophan amino acids showing the highest level of affinity. These findings offer valuable insights for the selection of compounds sharing a similar structure and possessing a high affinity for A42R, potentially capable of inhibiting its enzyme activity. The study highlights a structural advantage and paves the way for the development of targeted therapies against MPXV infections.

Potentially pathogenic culturable bacteria in hemodialysis waters

BackgroundHemodialysis patients are at risk of acquiring healthcare-related infections due to using non-sterile water to prepare hemodialysis fluid. Therefore, microbiological control and monitoring of used water are of crucial importance.Materials and methodsIn this work, we identified bacterial populations occupying a hemodialysis water distribution system for almost a 6-month period in Ahvaz city, southwest of Iran. A total of 18 samples from three points were collected. We found high colony counts of bacteria on R2A agar. 31 bacteria with different morphological and biochemical characteristics were identified by molecular-genetic methods based on 16 S rRNA gene sequencing. Endotoxin concentrations were measured, using Endosafe® Rapid LAL Single-Test Vials.ResultsA diverse bacterial community was identified, containing predominantly Gram-negative bacilli. The most frequently isolated genus was Sphingomonas. Five species including M. fortuitum, M. lentiflavum, M.szulgai, M. barrassiae, and M. gordonae was identified .Despite the presence of Gram-negative bacteria the endotoxin analysis of all samples revealed that their endotoxin values were below the detection limit.ConclusionThe members of Sphingomonas genus along with Bosea and mycobacteria could be regarded as pioneers in surface colonization and biofilm creation. These bacteria with others like Pelomonas, Bradyrhizobium, staphylococcus, and Microbacterium may represent a potential health risk to patients under hemodialysis treatment.

Acinetobacter nosocomialis utilizes a unique type VI secretion system to promote its survival in niches with prey bacteria.

Pathogenic bacteria of the Acinetobacter genus pose a severe threat to human health worldwide due to their strong adaptability, tolerance, and antibiotic resistance. Most isolates of these bacteria harbor a type VI secretion system (T6SS) that allows them to outcompete co-residing microorganisms, but whether this system is involved in acquiring nutrients from preys remains less studied. In this study, we found that Ab25, a clinical isolate of Acinetobacter nosocomialis, utilizes a T6SS to kill taxonomically diverse microorganisms, including bacteria and fungi. The T6SS of Ab25 is constitutively expressed, and among the three predicted effectors, T6e1, a member of the RHS effector family, contributes the most for its antimicrobial activity. T6e1 undergoes self-cleavage, and a short carboxyl fragment with nuclease activity is sufficient to kill target cells via T6SS injection. Interestingly, strain Ab25 encodes an orphan VgrG protein, which when overexpressed blocks the firing of its T6SS. In niches such as dry plastic surfaces, the T6SS promotes prey microorganism-dependent survival of Ab25. These results reveal that A. nosocomialis employs T6SSs that are highly diverse in their regulation and effector composition to gain a competitive advantage in environments with scarce nutrient supply and competing microbes.IMPORTANCEThe type VI secretion system (T6SS) plays an important role in bacterial adaptation to environmental challenges. Members of the Acinetobacter genus, particularly A. baumannii and A. nosocomialis, are notorious for their multidrug resistance and their ability to survive in harsh environments. In contrast to A. baumannii, whose T6SS has been well-studied, few research works have focused on A. nosocomialis. In this study, we found that an A. nosocomialis strain utilizes a contitutively active T6SS to kill diverse microorganisms, including bacteria and fungi. Although T6SS structural proteins of A. nosocomialis are similar to those of A. baumannii, the effector repertoire differs greatly. Interestingly, the T6SS of the A. nosocomialis strain codes for an ophan VgrG protein, which blocks the firing of the system when overexpressed, suggesting the existence of a new regulatory mechanism for the T6SS. Importantly, although the T6SS does not provide an advantage when the bacterium is grown in nutrient-rich medium, it allows A. nosocomialis to survive better in dry surfaces that contain co-existing bacteria. Our results suggest that killing of co-residing microorganisms may increase the effectiveness of strategies designed to reduce the fitness of Acinetobacter bacteria by targeting their T6SS.

The isolation and identification of Bacillus velezensis ZN-S10 from vanilla (V. planifolia), and the microbial distribution after the curing process

The market value of vanilla beans (Vanilla planifolia) is constantly increasing due to their natural aroma and flavor properties that improve after a curing process, where bacteria colonization plays a critical role. However, a few publications suggest that bacteria play a role in the curing process. Hence, this study aimed to isolate Bacillus sp. that could be used for fermenting V. planifolia while analyzing their role in the curing process. Bacillus velezensis ZN-S10 identified with 16S rRNA sequencing was isolated from conventionally cured V. planifolia beans. A bacteria culture solution of B. velezensis ZN-S10 (1 mL of 1 × 107 CFU mL−1) was then coated on 1 kg of non-cured vanilla pods that was found to ferment and colonize vanilla. PCA results revealed distinguished bacterial communities of fermented vanilla and the control group, suggesting colonization of vanilla. Phylogenetic analysis showed that ZN-S10 was the dominant Bacillus genus member and narrowly correlated to B. velezensis EM-1 and B. velezensis PMC206-1, with 78% and 73% similarity, respectively. The bacterial taxonomic profiling of cured V. planifolia had a significant relative abundance of Firmicutes, Proteobacteria, Cyanobacteria, Planctomycetes, and Bacteroidetes phyla according to the predominance. Firmicutes accounted for 55% of the total bacterial sequences, suggesting their colonization and effective fermentation roles in curing vanilla.

Comparative analysis of optional hunting behavior in Cricetinae hamsters using the data compression approach

Research into the hunting behavior in members of the Cricetidae family offers an opportunity to reveal what changes in the predatory behavioral sequences occur when a rodent species shifts from an omnivorous to a predatory lifestyle. The study tests the following hypotheses: are there phylogenetic differences in the divergence of species’ predatory lifestyles in hamsters or do ecological factors lead to shaping their hunting behavior? We applied the data compression approach for performing comparative analysis of hunting patterns as biological “texts.” The study presents a comparative analysis of hunting behaviors in five Cricetinae species, focusing on the new data obtained for the desert hamster Phodopus roborovskii whose behavior has never been studied before. The hunting behavior of P. roborovskii appeared to be the most variable one. In contrast, behavioral sequences in P. campbelli and Allocricetulus curtatus display more significant order and predictability of behavior during hunting. Optional hunting behavior in the most ancient species P. roborovskii displayed similarities with obligate patterns in “young” Allocricetulus species. It thus turned out to be the most advanced hunter among members of the Phodopus genus. Differences in hunting sequences among Phodopus representatives suggest that the hunting behavior of these species, despite its optional mode, was subject to selection during species splitting within the genus. These results did not reveal the role played by phylogenetic differences in the divergence of species’ predatory lifestyles. They suggested that ecological conditions are the main factors in speciation of the hunting behavior in hamsters.

The TolC and Lipopolysaccharide-Specific Escherichia coli Bacteriophage TLS-the Tlsvirus Archetype Virus.

TLS is a virulent bacteriophage of Escherichia coli that utilizes TolC and lipopolysaccharide as its cell surface receptors. The genome was reannotated using the latest online resources and compared to other T1-like phages. The TLS genome consists of 49,902 base pairs, encoding 86 coding sequences that display considerable sequence similarity with the T1 phage genome. It also contains 18 intergenic 21-base long repeats, each of them upstream of a predicted start codon and in the direction of transcription. Data revealed that DNA packaging occurs through the pac site-mediated headful mechanism. Based on sequence analysis of its genome, TLS belongs to the Drexlerviridae family and represents the type member of the Tlsvirus genus.

Tanapox Virus and Yaba Monkey Tumor Virus K3 Orthologs Inhibit Primate Protein Kinase R in a Species-Specific Fashion.

Yaba monkey tumor virus (YMTV) and Tanapox virus (TPV) are members of the Yatapoxvirus genus and can infect humans and other primates. Despite the threat posed by yatapoxviruses, the factors determining their host range are poorly understood. In this study, we analyzed the ability of YMTV and TPV orthologs of vaccinia virus K3 (called 012 in YMTV and TPV), which share 75% amino acid identity with one another, to inhibit PKR from 15 different primate species. We first used a luciferase-based reporter, and found that YMTV and TPV K3 orthologs inhibited PKR in a species-specific manner and showed distinct PKR inhibition profiles. TPV 012 inhibited PKR from 11 primates, including humans, substantially better than YMTV 012. In contrast, both K3 orthologs inhibited the other four primate PKRs comparably well. Using YMTV 012 and TPV 012 hybrids, we mapped the region responsible for the differential PKR inhibition to the C- terminus of the K3 orthologs. Next, we generated chimeric vaccinia virus strains to investigate whether TPV K3 and YMTV K3 orthologs could rescue the replication of a vaccinia virus strain that lacks PKR inhibitors K3L and E3L. Virus replication in primate-derived cells generally correlated with the patterns observed in the luciferase-based assay. Together, these observations demonstrate that yatapoxvirus K3 orthologs have distinct PKR inhibition profiles and inhibit PKR in a species-specific manner, which may contribute to the differential susceptibility of primate species to yatapoxvirus infections.

Robust Systemic and Mucosal Immune Responses to Coxsackievirus B3 Elicited by Spider Silk Protein Based Nanovaccines via Subcutaneous Immunization

AbstractCoxsackievirus B3 (CVB3) is a member of the enterovirus genus and linked to several diseases, including myocarditis, which can progress to dilated cardiomyopathy. Despite ongoing preclinical efforts, no clinically approved vaccines against CVB3 are currently available, highlighting the urgent need for effective prophylactic solutions. In this study, a nanovaccine platform based on spider minor ampullate silk protein (MiSp) is introduced. This platform utilizes protein nanoparticles engineered from chimeric proteins that incorporate CVB3 antigenic peptides into customized MiSp, subsequently loaded with all‐trans retinoic acid (RA). These functional nanovaccines are capable of eliciting both mucosal and systemic immune responses following subcutaneous administration and demonstrate significant protective effects against CVB3 infection in mice. This study signifies an approach in peptide‐based parenteral vaccine strategies, utilizing engineered MiSp nanoparticles combined with RA. This methodology represents a promising pathway for preventing enterovirus infections by leveraging the unique immunomodulatory properties of spidroins and RA to combat these pathogens effectively.

Discovery of a novel symbiotic lineage associated with a hematophagous leech from the genus Haementeria.

Obligate symbiotic associations with a nutritional base have likely evolved more than once in strict blood-feeding leeches. Unlike those symbioses found in hematophagous arthropods, the nature, identity, and evolutionary history of these remains poorly studied. In this work, we further explored obligate nutritional associations between Haementeria leeches and their microbial symbionts, which led to the unexpected discovery of a novel symbiosis with a member of the Pluralibacter genus. When compared to Providencia siddallii, an obligate nutritional symbiont of other Haementeria leeches, this novel bacterial symbiont shows convergent retention of the metabolic pathways involved in B vitamin biosynthesis. Moreover, the genomic characteristics of this Pluralibacter symbiont suggest a more recent association than that of Pr. siddallii and Haementeria. We conclude that the once-thought stable associations between blood-feeding Glossiphoniidae and their symbionts (i.e., one bacteriome structure, one symbiont lineage) can break down, mirroring symbiont turnover observed in various arthropod lineages.

Unraveling female mate choice in Schizocosa mccooki: The interplay of male mass and vibratory courtship

AbstractSexual selection is an important evolutionary force and despite extensive research, understanding mate choice on naturally occurring trait variation remains an intriguing area of study. The correlation between mass, a trait associated with courter viability and common target for mate choice, and signals is particularly noteworthy. This study focuses on Schizocosa mccooki, the largest member of a wolf spider genus renowned for its diverse complex male courtship. Our objective was to understand the relationship of male mass and other courtship signals on female choice within this species. We conducted lab experiments involving random pairings of field caught males and females, recording vibratory courtship and its outcomes. Our findings revealed that S. mccooki courtship consisted of vibratory signals with two major components, thumps and raps. Male mass, overall courtship vigor, and thump duration were found to predict mating success. Interestingly, we found no correlation between temporal traits and mass suggesting independent information and an absence of trade‐offs between mass, courtship vigor, and signal component rates/durations. Instead, we found that heavier males tend to produce thumps with lower frequencies. The finding that male mass predicts mating success contrasts with patterns observed in other species in this well‐studied genus. By uncovering the mate choice patterns of S. mccooki, this study underscores the importance of comparative studies to understand the ways in which sexual selection drives diversification, even among closely related taxa.

Characterization and Identification of Ipomoea genus in the Greater Kediri Area, Indonesia

This study aims to characterize and identify the genus of Ipomoea in the Greater Kediri area. The research was conducted in the Greater Kediri area which includes Kediri Regency and City, Nganjuk Regency, Blitar Regency and City, Tulungagung Regency, and Trenggalek Regency of East Java from January to June 2024 in an explorative descriptive manner. Sampling was done by snowball and purposive sampling. The observed characters include the morphological structure of roots, stems, leaves, flowers, fruits, and seeds. Plant identification was carried out referring to the Ipomoea genus Identification Key. The tools and materials used were ruler, razor/cutter, camera, stereo microscope, stationery, and plants of Ipomoea genus that were found. Ipomoea genus found in Greater Kediri area were 12 types, namely: Ipomoea batatas, Ipomoea carnea, Ipomoea aquatica, Ipomoea reptans, Ipomoea triloba, Ipomoea lacunosa, Ipomoea obscura, Ipomoea quamoclit, Ipomoea quinquefolia, Ipomoea pes-tigridis, Ipomoea pes-caprae and Ipomoea tricolor. Because of the wide area of research, this research is continued and if new species are found, they will be collected and added to the database of Ipomoea genus members in the Greater Kediri area.

A simplified single-leaflet graft inoculation method and a Duplex PCR assay for the identification of the Tomato Yellow Leaf Curl Virus infection in tomatoes

Tomato yellow leaf curl virus (TYLCV), a member of the Geminivirus genus, has caused an epidemic disease in tomato worldwide, including in Azerbaijan. TYLCV is a phloem-limited virus that is transmitted in a semi-persistent manner by whiteflies (Bemisia tabaci). Experimental propagation of TYCV has been performed primarily by using the single-leaflet grafting inoculation method. To develop a simple and effective method for transmission of TYLCV, we investigated grafting single-leaflets from tomato plants infected with TYLCV to recipient tomato seedlings. Fifty-one (51) tomato seedlings were tested and forty-eight (48) were grafted successfully with single-leaflets infected with TYLCV. Among them, forty-six (46) seedlings (92%) were systemically infected with TYLCV and developed typical symptoms. In our study, we found that single-leaflet grafting could provide a sufficient amount of inoculum for the transmission of TYLCV to the grafted tomato seedlings. The reported results regarding the development of the TYLCV inoculation and monitoring method and its application to study host plant-virus interaction showed that it could be used as an efficient alternative inoculation method.

Subclinical Ovine Gammaherpesvirus 2-Related Infections in Free-Ranging Wild Boars (Sus scrofa) from Southern Brazil.

Ovine gammaherpesvirus 2 (OvGHV2), is a Macavirus and the cause of sheep-associated malignant catarrhal fever (SA-MCF), in which sheep are the asymptomatic reservoir hosts. Susceptible mammalian populations infected by OvGHV2 may develop clinical SA-MCF or subclinical infections. All members of the Macavirus genus known to be associated with MCF are collectively referred to as the MCF virus (MCFV) complex. This report describes the occurrence of subclinical OvGHV2-related infections in free-ranging wild boars (Sus scrofa) from southern Brazil. Specific body organs (n = 14) and biological samples (nasal and oral swabs; n = 17) were collected from 24 asymptomatic wild boars from a conservation unit located within the Central-eastern mesoregion of Paraná State. Organs were processed to observe histopathological patterns suggestive of diseases of domestic animals; only pulmonary samples were used in an immunohistochemical assay designed to detect MCFV tissue antigens. Furthermore, all samples were submitted to molecular assays designed to detect the OvGHV2 tegument protein gene. Viral-induced pneumonia was diagnosed in two wild boars; one of these contained OvGHV2 DNA, with MCFV antigens identified in the other. Additionally, MCFV tissue antigens were detected within pulmonary epithelial cells of the lungs with and without pulmonary disease. Collectively, OvGHV2 was detected in 37.5% (9/24) of all wild boars, with detection occurring in the organs of 57.1% (8/14) wild boars and the oral cavity of one animal. These results demonstrated that these wild boars were subclinically infected by OvGHV2, and that infection produced typical pulmonary alterations. In addition, the detection of OvGHV2 within the oral cavity of one wild boar may suggest that this animal may be a potential disseminator of this pathogen to susceptible animal populations, including livestock and wildlife, acting as a possible bridge host for OvGHV2. Furthermore, infection by OvGHV2 probably occurred due to incidental contact with asymptomatic sheep maintained within the surrounding rural areas and not within the conservation units.

The Relationships of Closely Related Cossid Moth Species among Geographically Diverse with Respect to Indian Cossid Moth: (Lepidoptera: Cossidae)

This research article presents a molecular phylogenetic investigation focusing on closely related Cossid moth species within the Cossidae family from diverse geographical areas. The study aims to identify the species as well determine the evolutionary relationships among closely related Cossid moth species as many of them looks very similar and are difficult to distinguish only basing on morphological characters. We first amplified approximately 700 base pairs (bp) of the mitochondrial cytochrome c oxidase subunit-1 gene (MT-CO1), extracted from the thoracic region of the adult cossid moths which were collected from Kedima-Nagaland, India. Although, morphologically many Cossid species has been extensively studied, its evolutionary relationship to other members of the same genus, family or sub-families remains uncertain being a very diverse group. Therefore, this study provides the first description of the evolutionary relationship between the studied Cossid moth with respect to other geographically diverse Cossid species. To analyse the connections among the Cossid species, phylogenetic trees were constructed using data from an additional 58 Cossid species. The Maximum Likelihood and Bayesian analysis were used to construct the phylogenetic tree. In Both the techniques it demonstrated robust bootstrap support for the obtained phylogenetic relationships. Our study effectively identified the Cossid species and presented compelling evidence for the CO1 gene's efficacy in differentiating closely related species, as corroborated from our phylogenetic analysis. These results enhance our understanding of the evolutionary dynamics within the Cossidae family and offer valuable insights into the phylogenetic associations among closely related Cossid moth species. Here, we have also reported new MT-CO1 sequence of the studied Cossid species and have deposited in GenBank database [PP358253].

Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India

Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507 bp partial coat protein gene (pCPG) segment was amplified from infected kinnow leaf tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23 kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC-RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL−1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507 bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL−1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.