The present study documented the first record of Euphlyctis kalasgramensis from Punjab, Pakistan. The specimens were collected during field visits from June through August 2018. Various morphometric measurements of E. kalasgramensis were taken and compared with Euphlyctis cyanophlyctis. Snout-vent length (SVL) was 38.11 +/- 0.87 mm (n = 5), snout length was 3% of SVL, foot length was 55% of SVL, head length was 32% of SVL and weight was 8.01 +/- 0.12 g (n = 5). A few specimens (n = 2) were euthanized and preserved for molecular analysis through mitochondrial 16S rRNA gene sequences. The newly obtained DNA sequences of E. kalasgramensis were submitted to GenBank and accession numbers were obtained (MK881165.1 and MK920114.1). The Maximum likelihood and Neighbor-joining trees based on Kimura 2-parameter distance resulted in similar phylogenetic trees. Euphlyctis kalasgramensis was out group in both phylogenetic trees. The interspecific divergence of E. kalasgramensis and E. cyanophlyctis was high ranging from 4% to 6% as compared to low intraspecific divergence 0% and 1%. The diversity and distribution ranges of many amphibians species are not well known in Pakistan due to lack of taxonomic information. In our recommendation, a large scale DNA barcoding is required to report more cryptic or new species from Pakistan.

The Indus valley toad and common Asian toad are widely distributed toads in Pakistan. There is doubt in the taxonomic position of species within the genus Duttaphrynus in Pakistan as most of the species identified on morphology. Previously, Bufo melanostictus hazarensis identified on morphology but during the present study, it is confirmed as Duttaphrynus melanostictus-based COI sequences (MK941836). The interspecific divergence between Duttaphrynus stomaticus and D. melanostictus was 16%. The intraspecific divergence of D. stomaticus (MK947909.1) was ranging from 0% to 1% while the intraspecific divergence of D. melanostictus (MK941836) was high ranging from 10% to 11%. Overall, genetic variation between the species of genus Duttaphrynus based on p-distance was 14%. In our recommendation, a large-scale molecular identification of amphibians should take into consideration for exact species identification to report any new species from Pakistan.

The present study was conducted to assess the efficacy of Laboratory-Isolated Lactocaseibacillus casei NK1 (Lc. NK1) in broilers hypothesizing that, Lc. NK1 supplementation will enhance growth performance, modulate the gut microbiome, and reduce fecal pathogenic Escherichia coli in broilers. The experiment spanned 35 days where 60 one-day-old broiler chicks were randomly assigned to four treatment groups (n = 15); control-group with no treatment (NC), APEC (challenged with E. coli only), CProb (received commercial probiotics), and LNK1 (treated with Lc. NK1). The APEC, CProb, and LNK1 groups were infected with E. coli O78 strain at 11 days of age. Growth performance analysis revealed that the LNK1 group by day 35 gained body weight similar to the CProb group, with both groups significantly outperforming the APEC group (p < 0.001). Both the LNK1 and CProp groups exhibited similar reduction in E. coli while increasing Lactobacillus colorizations in the cloacal swabs from day 21 to 35 of age (p > 0.05). Metagenomic analysis using 16S rRNA sequencing showed that the LNK1 group maintained a diverse and balanced gut microbiota, characterized by increased Firmicutes and reduced Proteobacteria. In contrast, the APEC group exhibited reduced diversity and dominance of Escherichia-Shigella (p < 0.001). These findings suggest Lc. NK1 could be a promising probiotic for enhancing gut health and growth performance in broilers, even under pathogenic challenges, offering a potential alternative to antibiotics in poultry production.

The present study was planned to characterize the oral microbiome of Pteropus medius using n ext- g eneration s equen c in g and to evaluate its potential zoonotic implications. Six specimens were sampled from rural and urban sites namely Kasur, Okara and Lahore, Punjab-Pakistan. The comparative metagenomic analysis revealed significant differences in the microbial composition between rural and urban roosting sites. Bat s that were capture d from the rural sites ha d dominance of Proteoba c teria (81%) followed by Firmicutes (16%) and Actinobacteria (3%). The bacterial genera such as Escherichia (76%), Stre p tococcus (8%) and Staphylococcus (5%) were identified. In contrast, urban sampling sites showed lower relative abundance of Proteobacteria (68%) followed by Actinobacteria (9%). The bacterial genera includ ing Cellvibrio (51%), Sphingobact e rium (11%), and Stenotrophomonas (9%) were identified. The identification of pathogenic genera viz., Escherichia , Strept o coccus , and Staphylococcus highlights the potential zoonotic threat posed by Pteropus medius . It can be concluded that there is a need for continuous monitoring and assessment of the microbial communities in P . medius populations mostly in the areas with high human-bat interactions. The small sample size and restricted geographical scope is the limitation of the present study so future research with larger sample sizes and more diverse sites could provide a more detailed understanding of the environmental and ecological factors influencing the P. medius microbiome. Moreover, functional analysis of the identified bacterial taxa such as their role in metabolism, immunity and pathogen resistance could provide detailed insights into the health implications and help understand the mechanisms of pathogen transmission from bats to huma ns.

The present study was conducted in district Kasur, Pakistan to assess the guava fruit damage by Pteropus medius during summer and winter fruit ripening seasons in 2023-24. Artificial LED (AL) lights of 12W and 30W were used as a management strategy. Damage guava fruits by bats were collected for thirty days during both ripening seasons of guava (Psidium gujava) from three randomly selected acres in orchard, with and without artificial lights. After counting and weighing the collected damaged fruits, economic loss was calculated. The data indicated that on daily basis (WADF) of 1.97 ± 0.17 kg per acre (p < 0.0001) of actual fruit damage was observed in the winter season, while summer season faced 1.27 ± 0.09 kg per acre (p < 0.0001). Seasonal yield loss of 2.6% in summer and 16.14% in winter was estimated. The use of 12W and 30W LED lights as management strategy significantly reduced the fruit damage and economic losses. During winter season, 30WAL and 12WAL showed reduced damaged fruit per acre (0.36 ± 0.03 kg and 0.59 ± 0.05, respectively) as compared to orchard (4.97 ± 0.17 kg) with no artificial light (NAL). The yield loss (%) was significantly lower when the orchard was exposed to LED lights. The lowest yield loss was recorded with 30WALs during both summer (0.13 ± 0.01%) and winter (0.17 ± 0.01%) as compared to yield loss (1.30 ± 0.04%) in summer and (2.32 ± 0.08%) in winter with no artificial lights (NAL). The initial cost of light installation was considerable but the reduction in fruit loss made it economically worthwhile. In terms of cost-effectiveness, the 12WAL lights provided a higher return on investment, with a noteworthy positive net economic return during the winter season. It can be concluded that the installation of artificial lights is a practical and effective solution to manage bats related damage in guava orchards. The 12W AL lights offer a good balance between cost and effectiveness, making them a smart choice for orchard owners looking to minimize damage and maximize their profits without harming the bat population. Keywords: Artificial Lights, Indian Flying Fox, Pteropus medius, Fruit damage, Conservation.