Masood Ur Rehman¹, Syed Muhammad Suhail¹, Iqbal Munir², Rajwali Khan^1*

¹Department of Livestock Management, Breeding and Genetics, The University of Agriculture, Peshawar, 25130, Khyber Pakhtunkhwa, Pakistan

²Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, 25130, Khyber Pakhtunkhwa, Pakistan

Abstract

The current study aimed to evaluate the impact of three different agroecological zones on the productive and reproductive performance of Holstein-Friesian (HF) cows, along with their comparative analysis of physiological and molecular markers under subtropical conditions of Pakistan. The productive (milk yield and composition) and reproductive (service per conception and calving interval) performance of genetically identical (n=210) HF cows placed across three agroecological zones: irrigated lowlands (Okara), wet (Abbottabad), and dry (Queta) highlands were analyzed. Additionally, heat stress markers (superoxide dismutase, glutathione peroxidase, serum cortisol, bovine heat shock protein 70 (HSP-70), and blood glucose) from five cows in each location (n=15 in total) were also investigated in early and late summers using commercial ELISA and calorimetric kits. Furthermore, the mRNA levels of heat shock protein genes HSPA8 and HSP90AB1 were also quantified in three agroecological zones through qRT-PCR.  The results revealed that cows raised in the wet highlands of Abbottabad exhibited significantly higher (P<0.05) milk production (daily and total lactational yield), and the lowest service period, calving interval and number of services per conception as compared to the cows from the other two agroecological zones. Furthermore, there was a significant effect (P<0.05) of agroecological zones on the heat stress indicators (SOD, GPX, cortisol, blood glucose, and HSP70) profiles and expression of heat shock protein genes in HF cows. The dairy cows from Abbottabad (Wet highlands) showed a significantly lower profile of heat stress indicators as compared to the cows from the other two agroecological zones. It can be inferred that HF cows function better in moderate agroecological zones of subtropical countries.

Keywords: Heat stress indicators, Subtropics, Holstein-Friesian cows, Agroecological zone

Heer Baloch¹, Irfan Ali Sabir², Saadullah Khan Leghari¹, Muhammad Sohail Saddiq^3*, Pravej Alam⁴, Shahbaz Khan^5*, Esha Mehik Fatima⁶, Mateen Sajid⁷, Muhammad Hammad Raza⁸, Muhammad Arif Hussain¹, Muhammad Ayoub¹, Rashid Iqbal⁹

¹Department of Botany, Ghazi University, Dera Ghazi Khan, Pakistan

²College of Horticulture, South China Agricultural University, Guangzhou, 510642, China

³Department of Agronomy, Ghazi University, Dera Ghazi Khan, Pakistan

⁴Department of Biology, College of Sciences and Humanities, Prince Sattam Bin Abdulaziz University, Al- Kharj 11942, Saudi Arabia

⁵Colorado Water Center, Colorado State University, Fort Collins, CO, USA

⁶Department of Entomology, Muhammad Nawaz Shareef University of Agriculture, Multan, Pakistan

⁷Department of Horticulture, Ghazi University, Dera Ghazi Khan, Pakistan

⁸Department of Agri. Extension and Education, Ghazi University, Dera Ghazi Khan, Pakistan

⁹Department of Agronomy, Faculty of Agriculture and Environment, The Islamia University of Bahawalpur, Pakistan

Abstract

Moringa leaf extract (MLE) as a biostimulant has demonstrated success in boosting the productivity of several agronomic crops, but its impact on Buckwheat crops remains unexplored. Buckwheat, recognized as an essential nutritional and functional food crop, often exhibits lower yields compared to major cereal crops grown in similar environments. Therefore, this research aimed to investigate the impact of different concentrations (1%, 2%, and 3%) of MLE on the agricultural performance of common buckwheat (CB) and tartary buckwheat (TB). A pot experiment was carried out according to completely randomized design with factorial arrangements having three replications. Results demonstrated significant improvements in growth parameters (branches, leaves, nodes, and internodes) for MLE-treated plants compared to the control group. Foliar treatment MLE 2% also increased chlorophyll content, improved membrane stability index (MSI) and relative water content (RWC), and enhanced biochemical composition (phenolic compounds, free amino acids, leaf proline, and soluble sugars) in both buckwheat genotypes followed by MLE 3% and MLE 1%. TB produced significantly higher grain yield (0.74 g) as compared to CB (0.43 g). The findings showed that a foliar treatment of MLE 2% led to increased grain yield in both TB (0.97 g) and CB (0.55 g) as compared to control group plants (0.37g TB and 0.22 g CB) respectively. This increase was associated with elevated activities of photosynthetic pigments, phenolic content, RWC, free amino acids, soluble sugars, and catalase in both buckwheat genotypes. In conclusion, MLE application at 2% significantly boosted the agriculture performance of buckwheat, and this study unlocked new insights into optimizing the productivity of the vital food crop.

Keywords: Biostimulant, Free amino acid, Phenolic, Buckwheat, Foliar application, Grain yield

Aeman Jilani¹, Mirza Imran Shahzad^1*, Muhammad Mohsin Zaman¹, Areeba Yousaf¹, Gildardo Rivera²

¹Institute of Biochemistry, Biotechnology and Bioinformatics, The Islamia University of Bahawalpur, Pakistan

2Laboratorio de Biotecnología Farmacéutica, Centro de Biotecnología Genómica, Instituto Politécnico Nacional, México

Abstract

Tuberculosis (TB), the white plague of Europe is still uncontrolled and fatal in many parts of the world including Pakistan. It is a major cause of morbidity and mortality in human and domestic animals in Pakistan. No new vaccine in the last hundred years has been developed except for a few encouraging results from recombinant and DNA vaccines in the past two decades. Five Mycobacterium specific genes (Rv0379, Rv3914, Rv3006, Rv0432+SP, and Rv0432-SP) were selected to develop DNA vaccine(s). All the constructs were tested on mice using both naked DNA and prime-boost methodologies. Forty-five BALB/c mice were divided into three main groups; DNA vaccine group, BCG Prime boost group, and Control group. Post-vaccine (PV) and post-challenge (PC) immune responses were confirmed through cytokine ELISA and qRT PCR. IFN-γ was additionally checked in plasma as well. Based on cytokine ELISA PC immune responses showed significant differences in TNF-α levels for both naked DNA vaccine groups (Rv0379, Rv3006, and Rv0432-SP) and BCG primed Rv3914 group in comparison to the BCG control group (p<0.05). Based on qRT PCR, IL-6, TNF-α, IFN-γ, and IL-1β showed no significant difference among all the vaccines and BCG control groups (CT range 25- 30). IFN-γ levels in plasma were analyzed PC; two vaccines Rv3006/LppZ and BCG primed Rv0432/SodC-SP (highest mean value 1360.35 pg/ml) have shown significant results (cutoff value 21pg/ml) at 63 days. All the vaccine construct(s) alone or in combination have significant therapeutic effects in comparison to BCG and negative control groups.

Keywords: Mycobacterium tuberculosis, DNA vaccine, Tumor necrosis factor-alpha, Interleukin-6, Interferon-gamma, Interleukin-1beta, Bacille Calmette-Guérin (BCG)

Tayyaba Asghar¹, Mudassar Mohiuddin^1*, Ayesha Mohiud Din^2,3, Tahira Kamal⁴, Muhammad Khalid Mansoor¹,

Abubakar Siddique^5,6,7, Mudasser Habib⁸, Riaz Hussain⁹, Muhammad Taslim Ghori¹⁰, Hira Hameed¹, Humaira Rizwana¹¹,

Islem Abid¹², Muhammad Ehsan¹³, Aroosh Shabbir¹⁴

¹Department of Microbiology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

²Department of Biotechnology, Virtual University of Pakistan, Lahore, Pakistan

³School of Biological Sciences, University of Southampton, Southampton, UK

⁴National Institute for Genomics and Advanced Biotechnology, National Agricultural Research Centre (NARC), Islamabad, Pakistan

⁵Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Science and Technology (NUST), Islamabad, Pakistan

⁶Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, China

⁷Hainan Institute of Zhejiang University, Sanya, China

⁸Animal Sciences Division, Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan

⁹Department of Pathology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

¹⁰Department of Clinical Medicine and Surgery, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

¹¹Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

¹²Centre of Excellence in Biotechnology Research, King Saud University, P.O Box 2455, Riyad 11495, Saudi Arabia

¹³Department of Parasitology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

¹⁴Institute of Molecular Biology and Biotechnology (IMBB), The University of Lahore, Pakistan

Abstract

The lumpy skin disease virus (LSDV) is a member of the Capripox genus of the Poxviridae family. It is the causative agent of lumpy skin, a highly contagious disease of cattle, water buffalo, sheep, and goats. In 2022, several outbreaks of LSD were reported in the Cholistan region of Pakistan, which has a large population of livestock living in arid conditions. A total of 230 blood/serum and scab samples were collected from three LSD outbreak locations. Clinically, affected cattle showed acute clinical signs characterized by skin nodules, fever, enlarged lymph nodes, emaciation, and lower leg edema. Hematological findings revealed non-significant changes in red blood cell and white blood cell counts (some animals had leukocytosis while others were leukopenic) whereas, hemoglobin level were significantly low. Platelet count, MPV, PCT, P-LRC, and P-LCC were elevated. Granulocytes were significantly low in LSD affected cattle while lymphocyte counts were significantly high. Serological findings revealed elevated protein levels, along with high creatinine and ALT concentrations. Amplification of DNA-dependent RNA polymerase 30 kDa subunit gene (RPO30) confirmed the presence of LSD virus in all suspected samples. Phylogenetic analysis showed that all Pakistani isolates clustered closely with isolates from neighboring countries. The SNPs differences were less than 20 among these isolates, indicating their close resemblance with each other. It can, therefore, be inferred that our LSD strains might be originated from neighboring Asian countries, that were affected by LSD in previous years.

Keywords: Lumpy skin disease virus (LSDV), Cholistan, Skin lesions, Hematology, Phylogenetic analysis

Pozdnyakova Yelena^1*, Solyanov Dmitry², Tatina Yelena¹, Britko Valeriy¹, Omarbekova Nazgul³, Korshukova Marina³

¹Department of Biomedicine, Karaganda Medical University, Karaganda, The Republic of Kazakhstan

²Department of Pharmacy, Karaganda Medical University, Karaganda, The Republic of Kazakhstan

³Department of Informatics and Biostatistics, Karaganda Medical University, Karaganda, The Republic of Kazakhstan

Abstract

The aim of this study is to investigate the effects on the nervous system of the use of Stachys sieboldii root powder, in growing male rats on a high fat and sucrose (HFHS) diet. The animals were divided into three groups: Intact – normal rodent chow, HFHS – high fat and sucrose diet, HFHS + Stachys – high fat and sucrose diet with Stachys supplementation. After 30 days of the experiment, the animals were subjected to the “Open Field” test to study the changes in behavioural responses. Biochemical studies of changes in the concentration of lipid peroxidation products in brain homogenates of growing rats were also carried out. Growing males on the HFHS diet exhibit altered nervous system functioning. Animals show apathy, their motor activity and indicators of exploratory behaviour are reduced and the level of emotional reactivity increases. On the part of biochemical changes – the level of all indicators of lipid peroxidation increases significantly. The introduction of Stachys root powder reduces the negative effect of the diet on the organism of animals – they significantly increase the indicators of exploratory behaviour, increase locomotor activity and reduce emotional reactivity, as well as there is a decrease in the concentration of all products of free-radical oxidation in the brain homogenate. Our study showed that diets high in fat and sucrose had negative effects on growing male rats. The introduction of Stachys sieboldii root powder into the diet improved both behavioural response scores and antioxidant processes in the brain.

Keywords: Stachys sieboldii, Open field, Lipid peroxidation, Conjugated dienes, Ketodienes

Maria Rasool, Muhammad Hidayat Rasool^*, Mohsin Khurshid, Bilal Aslam

Institute of Microbiology, Government College University Faisalabad, Faisalabad-38000, Pakistan

Abstract

Multi-drug resistant (MDR) bacterial infections significantly increase mortality, morbidity, and treatment costs when they persist. Therefore, there is a pressing need to discover, modify, or search for antimicrobial agents with the ability to combat MDR bacteria. Silver nanoparticles used in this study were synthesized by Bacillus subtilis and characterized through different techniques. MDR strains underwent antibacterial activity, antioxidant activity, and time-kill kinetic assays to assess susceptibility to silver nanoparticles. Furthermore, the synergistic impact of silver nanoparticles and antibiotics was examined using the two-dimensional checkerboard method to calculate the Fractional Inhibitory Concentration Index (FICI). Scanning Electron Microscopy (SEM) results revealed a circular shape of synthesized AgNPs, with an average length and area of 76.78 and 47.10 nm, respectively. UV analysis showed an optimum peak at 420 nm. XRD analysis indicated the crystalline nature of nanoparticles with diversity in size. Remarkable antioxidant potential (55% of AgNPs) was observed at a concentration of 1000 µg/ml, while minimum activity (18%) was noted at 62.6 µg/ml. Silver nanoparticles demonstrated a synergistic interaction with the antibiotic Cefixime against Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae, with FICI values of 0.37, 0.3, 0.25, and 0.49, respectively, and an additive effect against Pseudomonas aeruginosa, with a FICI value of 0.7. Moreover, this research explores the anti-inflammatory potential of silver nanoparticles (AgNPs) in two distinct models: formaldehyde-induced inflammation and carrageenan-induced inflammation, along with an assessment of their in vitro anti-inflammatory activity. The findings shed light on the multifaceted role of AgNPs in mitigating inflammatory responses, offering promising avenues for therapeutic interventions.

Keywords: Green synthesis, Silver, Bacillus subtilis, Synergistic effect, Cefixime, XRD

Urairat Mongmonsin¹, Prapenpuksiri Rungsa², Withan Teajaroen¹, Theerasak Somdee³, Jureerut Daduang⁴, Sakda Daduang^2,5*

¹Department of Biomedical Sciences, Faculty of Graduate School, Khon Kaen University, Khon Kaen, Thailand

²Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, Thailand

³Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand

⁴Department of Clinical Chemistry, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand

⁵Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand

Abstract

Cordyceps militaris has been used in traditional medicine due to its immune-boosting functions, especially in elderly and cancer patients. The study aims to assess the impact of C. militaris extract on the immune response in LPS-induced splenocytes and male Sprague Dawley rats. The study found that CME contains various bioactive compounds such as adenosine, cordycepin (3′ deoxyadenosine), and phenolic compounds. In vitro studies showed that CME has the potential to enhance CD3⁺ T cell and CD45RA⁺ B cell populations in LPS-induced splenocytes with a cell viability of over 80%. Moreover, we found that CME can enhance the immune response in rats by increasing CD3⁺ T cell proliferation and up-regulating IL-4, IL-6, and IL-17a expression, without affecting the rat’s body weight. The findings suggest that CME could be a promising immune enhancer for patients with weakened immune systems. However, further animal experiments with varying doses are essential to determine the optimal dosage for the successful development of CME as a therapeutic agent.

Keywords: Cordyceps militaris, Immunostimulatory effects, Pro-inflammatory cytokines, Cordycepin

Jian Huang¹, Xiaohua Chen¹, Qinglian Zhong^1*

[1] Department of Gastroenterology, The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, Guangdong 518033, China

Abstract

The dysbiosis of the gut microbiota caused by drug metabolism and diets may influence the gastrointestinal (GI) barrier and their ability for normal attachment and further immunity system, which all could be associated with the medical efficacy of colorectal cancer (CRC) during chemotherapy.

This study aims to investigate Trimethylamine N-oxide (TMAO), a gastrointestinal product readily entering the bloodstream, as a potential risk factor for various diseases, including CRC.

To investigate the relationship between gut microbiota dysbiosis and carcinogenesis in CRC patients, we analyzed taxonomic alterations in the gut microbiota of 77 subjects, including 36 CRC patients and 41 normal controls. We collected samples of the participants’ microbiome from their fecal material and utilized 16S rRNA sequencing to identify the microbial composition. Additionally, to predict the functions of the GI microbiota, Phylogenetic Investigation of Communities by Reconstructing Unobserved States (PICRUSt) was employed. This could serve as a promising biomarker for colorectal cancer. Moreover, the serum level of TMAO between the CRC and healthy controls was also compared, and it was observed that the intestinal microbiome was changed in CRC patients; however, the serum level of TMAO was not correlated with the progression of CRC.

In conclusion, in the present study, instead of relying solely on TMAO, which is a convenient clinical test, we focused on the treatment of CRC by emphasizing the modification of the intestinal microbiome.

Keywords: CRC, Intestinal microbiome, TMAO, 16s rRNA, PICRUSt

Muhammad Amjad Iqbal^{1, 2}, Noreen Sarwar^1*, Sohail Raza¹, Sehrish Firyal³, Rabia Riaz⁴, Afia Muhammad Akram⁵, Rashad Munir⁴, Aamir Riaz Khan⁴, Mobeen Sarwar⁶, Muhammad Imran Arshad⁷

¹Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan

²Veterinary Research Institute, Lahore, Pakistan

³Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan

⁴Foot and Mouth Disease Research Centre, Lahore, Pakistan

⁵Department of Zoology, Division of Science and Technology, University of Education, Lahore, Pakistan

⁶Livestock and Dairy Development, Government of Punjab, Lahore, Pakistan

⁷Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan

Abstract

Inactivation of foot-and-mouth disease virus (FMDV) with Formalin (FA) and binary ethyleneimine (BEI) is a slow process and takes long time. The purpose of this study was to evaluate β-Propiolactone (BPL), an alkylating agent that is frequently used in vaccine development and production, as a candidate for the inactivation of FMDV serotype O. Virus was grown on confluent monolayer of BHK-21 cell line. Harvesting was performed between 18 and 20 hours after infection, when CPE was between 90 and 95%. The virus was subjected to inactivation with formalin (0.02%), BEI (2mM, 2.5mM, 3mM) and BPL (0.1%, 0.2 and 0.4%) at 4°C and 37°C. Samples (05 mL/each) were collected after 0, 2, 4, 8, 16, and 24 hours post treatment. The biological titer (TCID50/ml) of each inactivated sample was measured. An innocuity test was used to further confirm the inactivation. RT-PCR was used to detect viral genome damage by amplification of VP1 gene. Data were analyzed by one-way analysis of variance and pair wise comparison was made by post hoc Tukey’s HSD test. Linear regression was used in Microsoft Excel Version 2010. The virus titers after 0.02% formalin treatment were 7.172±0.298 and 4.584±1.362 at 4°C and 37°C respectively. The virus titers were 6.036±0.513, 5.622±0.298, and 5.150±0.449 with 2mM, 2.5mM, 3mM BEI at 4°C and 1.646±1.0210, 1.050±0.644, 0.492±0.492 at 37°C respectively. Inactivation with 0.1%, 0.2% and 0.4% BPL at 4°C resulted in virus titers of 2.386±1.1104, 1.4400±0.9445 and 0.6960±0.6960 respectively. Rapid inactivation of virus with all three BPL concentrations at 37°C gave mean titer of 0.00±0.00. There was a significant difference (P<0.05) among all treatment groups with highest inactivation rates recorded for BPL.

Keywords: FMDV, BHK-21, Inactivation, BEI, BPL, Formalin

Imran Bodlah^1*, Ammara Gull E Fareen², Umer Ayyaz Aslam Sheikh³, Muhammad Adnan Bodlah⁴, Rehana Bibi⁵, Hesham F. Alharby^{6, 7}, Habeeb M. Al-Solami⁶, Naser A. Alkenani⁶, Abdullah G. Al-Ghamdi⁶

¹Insect Biodiversity and Conservation Group, Department of Entomology, PMAS Arid Agriculture University, Rawalpindi, Pakistan

²Department of Environmental Sciences, PMAS Arid Agriculture University, Rawalpindi, Pakistan

³Department of Entomology, University of Poonch, Rawalakot, Azad Jammu and Kashmir, Pakistan

⁴Fareed Biodiversity Conservation Centre, Department of Agricultural Engineering, Khawaja Fareed University of Engineering and Information Technology, Rahim Yar Khan, Punjab, Pakistan
⁵Department of Plant Protection, Faculty of Agricultural Sciences, Ghazi University D. G. Khan

⁶Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia

⁷Plant Biology Research Group, Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia

Abstract

Bumble bees are among the insects that play a significant role in the pollination of several agricultural crops, fruits, vegetables, and wild flowering plants. Climate change has been discussed as a prospective threat to the biodiversity of these pollinators in different parts of the planet during the next several decades. As a result of expected climate change circumstances, a number of studies have determined that the distribution range of bumble bees will alter, with some species becoming extinct and majority of them relocating to the higher mountainous areas. Numerous countries around the globe have developed conservation measures for these ecologically and economically significant organisms. In Pakistan, Bombus haemorrhoidalis (Hymenoptera: Apidae) is more likely to be used as a pollinator in greenhouse vegetable production, as compared to other documented species. Our research aimed to find moderately and highly favorable locations for B. haemorrhoidalis in 2050 and 2070 based on two representative concentration pathways (RCP4.5 and RCP8.5) using Maxent. Five key contributing variables including Bio1 (Annual Mean Temperature), Bio12 (Annual Precipitation), Bio14 (Precipitation of Driest Month) and Bio18 (Precipitation of Warmest Quarter) along with 52 occurrence record of the species were utilized in the modeling procedure for determining potential distribution of B. haemorrhoidalis. Performance of the model was assessed by calculating the area under the curves (AUC), the partial ROC, the omission rates (E=5%), and the AICc (Model complexity). Regularization multiplier of finally selected model was 2. Based on the results of the Jackknife test, it was determined that only four climatic factors, namely Bio4, Bio12, Bio14 and Bio18 contributed 89.0% to the prediction of the species’ prospective distribution. The results indicated that highly suitable distribution areas of this species would be concentrated in upper the mountainous areas of Pakistan under the influence of climate change. The suitability of its habitat, however, will decrease under the forecasted climatic conditions of future scenarios (RCP 4.5 and RCP 8.5) for 2050 and 2070. According to the findings, the area that is anticipated to be moderately suitable will shrink by 320.492083 km²between the years 2050 and 2070 under RCP 4.5 (70), as compared to RCP 4.5. (50). In the same pattern, according to RCP 8.5 (70), it would shrink by a total of 260.764698 km² as compared to RCP 8.5. (50). A similar pattern would be observed for forecasted highly suitable areas, which would shrink by a total of 2492.820215 km² under RCP 4.5. (70) compared to RCP 4.5 (50), and by a total of 1363.441658 km² under RCP 8.5 (70) in contrast to RCP 8.5. (50). Results indicated that suitable areas for this species would decrease during two scenarios of year 2050 and 2070. This species would leave many areas of its current distribution under the influence of climate change and move upward towards upper mountainous areas of Azad Kashmir, Gilgit Baltistan and Khyber Pakhtunkhwa. Based on the results of our studies, Government along with other stakeholders of bee pollination may develop climate mitigation strategies to conserve pollination services of B. haemorrhoidalis on a sustainable basis. More extensive surveys are needed along with other tools of remote sensing for more reliable predictions.

Keywords: Bombus haemorrhoidalis, Habitat suitability, Environmental predictors, Maxent, Climate change, Pakistan