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The global seawater temperature is expected to further rise in the following years. While species have historically adapted to climatic variations, the current pace of climate change may exceed their ability to adapt. The abnormally increased seawater temperatures occasionally lead to high mortalities of marine bivalve mollusks, threatening the productivity of aquaculture and the sustainability of wild populations. This study investigates the antioxidant and cell death mechanisms of the Mediterranean mussel Mytilus galloprovincialis during a 25-day exposure to temperatures of 24°C, 26°C, and 28°C, by analyzing the transcription of key genes and assessing the oxidative damage on days 1, 3, 12, and 25. In addition, individuals resilient (survived at 28°C until day 30) and susceptible (died early at 26°C and 28°C) to thermal stress were collected to investigate potential polymorphisms in associated genes. The results showed increased transcription of antioxidant genes at higher temperatures. Elevated pro-apoptotic indices were initially observed at 26°C and a higher mortality than at 28°C. However, final mortality was much higher at 28°C. At 26°C, mussels exhibited the highest oxidative damage and pro-apoptotic indices after 25 days. At 28°C, although oxidative damage occurred after 24 hours, survivors maintained a prolonged activated antioxidant defense and increased lc3b transcription, which likely contributed to the observed reduction of pro-apoptotic and oxidative damage metrics on day 25, compared to 26°C. Further, the coding sequences of catalase , intracellular Cu-Zn superoxide dismutase ( Cu-Zn sod ), and fas-associated protein with death domain ( fadd ) from heat-resilient and heat-susceptible mussels were analyzed. Based on statistical correlation of nucleotide and genotype frequencies with resilience phenotypes, two novel single nucleotide polymorphisms (SNPs) in Cu-Zn sod and one in fadd were detected, potentially correlating with thermal stress resilience. These findings offer valuable insights into the physiological and genetic adaptations of M. galloprovincialis to rising temperatures and highlight loci potentially linking to thermal resilience.

Ectotherms are exposed to a range of environmental temperatures and may face extremes beyond their upper thermal limits. Such temperature extremes can stimulate aerobic metabolism toward its maximum, a decline in aerobic substrate oxidation, and a parallel increase of anaerobic metabolism, combined with ROS generation and oxidative stress. Under these stressful conditions, marine organisms recruit several defensive strategies for their maintenance and survival. However, thermal tolerance of ectothermic organisms may be increased after a brief exposure to sub-lethal temperatures, a process known as \"hardening\". In our study, we examined the ability of M. galloprovincialis to increase its thermal tolerance under the effect of elevated temperatures (24, 26 and 28 °C) through the \"hardening\" process. Our results demonstrate that this process can increase the heat tolerance and antioxidant defense of heat hardened mussels through more efficient ETS activity when exposed to temperatures beyond 24 °C, compared to non-hardened individuals. Enhanced cell protection is reflected in better adaptive strategies of heat hardened mussels, and thus decreased mortality. Although hardening seems a promising process for the maintenance of aquacultured populations under increased seasonal temperatures, further investigation of the molecular and cellular mechanisms regulating mussels’ heat resistance is required.

Freshwater crayfish Pontastacus leptodactylus is an indigenous European species of considerable ecological and commercial significance. However, it is highly susceptible to the oomycete Aphanomyces astaci , the causative agent of crayfish plague. Although its importance is widely recognized, little information exists on how seasonality affects the physiology and welfare of this species. The present study was designed to (i) investigate seasonal effects on the physiological responses of P. leptodactylus in relation to abiotic factors and crayfish plague outbreak, and (ii) identify the most suitable period for restocking in temperate regions. To achieve this, we characterized for the first time the mRNA expression of hsp70, hsp90, hif-1α, ubiquitin, and gapdh (as endogenous control gene), alongside protein levels of Hsp70, Hsp90, hydroxylated Hif-1α, ubiquitin conjugates, cleaved caspases, Bax/Bcl-2 ratio, and interleukin-6 (IL-6). Our findings revealed that cold stress triggered both the heat shock response and apoptosis. Interestingly, despite the activation of apoptosis, which generally suppresses inflammation, IL-6 levels increased, suggesting a possible association with A. astaci infection. Moreover, elevated Hif-1α levels indicated hypoxic stress during warmer months, likely linked to reduced oxygen availability and turbidity. Overall results suggest that crayfish plague epidemics may intensify with rising temperatures, whereas winter appears to be the optimal period for restocking. Interestingly, in addition, freshwater crayfish in temperate latitudes are organisms that face both cold and heat stress seasonally in the same ecosystem.

Pakistan has a huge sheep population (37.2 million in 2024) that is largely unexplored for the presence of vector transmitted parasites. Present study was aimed to document the prevalence of Anaplasma sp. , Anaplasma ovis , Theileria ovis and Theileria lestoquardi in sheep blood samples (N = 329) that were collected from six districts (Muzaffargarh, Rajanpur, Dera Ghazi Khan, Layyah, Taunsa and Khanewal) during August till December 2024 and to report the genetic diversity of screened pathogens. Molecular analyses revealed that the prevalence of Anaplasma sp. , Anaplasma ovis and Theileria ovis in screened sheep was 11%, 20% and 21% respectively. None of the screened sheep was Theileria lestoquardi infected. Co-infection of the screened pathogens was also observed. Presence of the detected pathogens was confirmed by DNA sequencing and subsequent BLAST analysis. Phylogenetic analysis revealed that these pathogens displayed genetic similarities with the sequences that were deposited from various countries across the globe. Prevalence of all screened pathogens varied significantly between the sampling districts. Similarly, the Anaplasma sp., Anaplasma ovis and Theileria ovis prevalence varied significantly among the sheep breeds. Anaplasma ovis infection was more common in large herds and in un-infested sheep. Theileria ovis infection was more frequent in small herds. In conclusion, we are reporting the presence of Anaplasma sp., Anaplasma ovis and Theileria ovis in Pakistani sheep that were enrolled from all six districts. Large-scale studies are recommended in various geo-climatic regions of Pakistan to confirm the genetic diversity, epidemiology and host-pathogen interactions that will contribute towards effective control of these infections among the local sheep population.

The members of genus Moniezia are the common parasites of livestock in tropical areas. The tapeworm, Moniezia expansa is commonly found in the gastrointestinal tract of the small and large ruminants. The present study focused on reporting the prevalence of M . expansa in small ruminants of southern Punjab: sheep and goats, in relation with epidemiological factors like age and gender. An overall prevalence of 27.2% was estimated for the small ruminants with higher infection rates in males (29.8%) and younger age group (<1 year; 32.9%). Moreover, the molecular characterization and phylogenetic analysis of the isolates based on partial cox1 gene indicated the placement of these sequences in the M . expansa cluster. Two distinct haplotypes, without any host tropism, were identified within the Pakistani isolates. A meta-analysis for M . expansa was run for all available global reports exhibiting an overall pooled prevalence of 21.3% (CI 95%: 13.5–29.0). Additionally, a global dataset encompassing 59 partial cox1 sequences submitted from different geographical locations was also assessed. Moderate haplotype diversity (0.760 ± 0.051) and significantly negative deviations from neutrality were estimated. The median joining haplotype network for these sequences revealed an interesting population structure indicating highly divergent sequences from China and Iraq compared to Pakistan, India, Vietnam, Senegal and Ethiopia. Given inconsistencies in genetic data there is a dire need to carry out molecular studies across the entire distributional range of M . expansa to delineate genetic diversity and population structure of the species. This will also be crucial in reevaluating the taxonomy of genus Moniezia .

Aquaculture is affected by numerous factors that may cause various health threats that have to be controlled by the most environmentally friendly approaches. In this context, prebiotics, probiotics, and synbiotics are frequently incorporated into organisms’ feeding rations to ameliorate the health status of the host’s intestine, enhancing its functionality and physiological performance, and to confront increasing antimicrobial resistance. The first step in this direction is the understanding of the complex microbiome system of the organism in order to administer the optimal supplement, in the best concentration, and in the correct way. In the present review, pre-, pro-, and synbiotics as aquaculture additives, together with the factors affecting gut microbiome in crayfish, are discussed, combined with their future prospective outcomes. Probiotics constitute non-pathogenic bacteria, mainly focused on organisms’ energy production and efficient immune response; prebiotics constitute fiber indigestible by the host organism, which promote the preferred gastrointestinal tract microorganisms’ growth and activity towards the optimum balance between the gastrointestinal and immune system’s microbiota; whereas synbiotics constitute their combination as a blend. Among pro-, pre-, and synbiotics’ multiple benefits are boosted immunity, increased resistance towards pathogens, and overall welfare promotion. Furthermore, we reviewed the intestinal microbiota abundance and composition, which are found to be influenced by a plethora of factors, including the organism’s developmental stage, infection by pathogens, diet, environmental conditions, culture methods, and exposure to toxins. Intestinal microbial communities in crayfish exhibit high plasticity, with infections leading to reduced diversity and abundance. The addition of synbiotic supplementation seems to provide better results than probiotics and prebiotics separately; however, there are still conflicting results regarding the optimal concentration.

Camel anaplasmosis is a tick-borne disease of zoonotic concern, yet its epidemiology in Pakistan remains understudied. This study aimed to determine the prevalence, associated risk factors, and phylogenetic characteristics of Anaplasma spp. in camels across diverse agro-climatic zones of Punjab. A total of 400 blood samples were collected from two districts—Jhang and Bahawalpur (n = 200 each)—using a multistage cluster sampling approach. From each district, four tehsils were selected; ten herds per tehsil were sampled, with five camels per herd. The PCR targeting the 16S rRNA gene was used for Anaplasma detection. Epidemiological data were gathered using a structured questionnaire. The overall prevalence was 25.75%. Multivariable analysis identified age (>5 years), district (Jhang), intensive management, and health status as significant risk factors. Phylogenetic analysis revealed that A. phagocytophilum isolates were genetically related to strains from India, Iran, and Turkey; A. platys showed proximity to dog-derived isolates from India, South Africa, and Spain; while Candidatus A. camelii was closely related to camel isolates from Egypt, China, Kenya, and Iran. In conclusion, camel anaplasmosis is prevalent in Punjab. Further research is warranted to explore the pathogenic potential and vector dynamics of circulating strains to devise control strategies.

Despite a goat population of approximately 80 million in Pakistan during 2020−2021, the prevalence of vector-borne pathogens in goats remains largely underexplored. This study aimed to assess the molecular prevalence and phylogenetic characteristics of Anaplasma ovis, Anaplasma marginale and Anaplasma phagocytophilum in goat blood samples (N = 239) collected from three districts (Muzaffargarh, Rajanpur, and Dera Ghazi Khan) in Punjab between September 2023 and October 2024. Blood samples were first screened with generic and then with species specific primers. Molecular analyses revealed a prevalence of 39% for Anaplasma spp. and 14% for A. ovis . A. marginale and A. phagocytophilum were not detected. DNA sequencing, by targeting 16S rRNA and msp4 genes, and BLAST analysis confirmed the presence of Anaplasma spp. and A. ovis, respectively. For both screening, bacterial prevalence rates varied significantly across sampling sites (P = 0.01 for Anaplasma spp. and P = 0.04 for A. ovis ). Additionally, the prevalence of Anaplasma spp. significantly differed among goat breeds (P = 0.004), while no association was found between goat sex and bacterial infections (P > 0.05 for both screening). Notably, Anaplasma spp. infection was associated with a significant decrease in red blood cell count and hemoglobin concentration, while A. ovis infection did not affect the complete blood count profile. Phylogenetic analysis revealed that our Anaplasma spp. isolates clustered with those from Iran, Cyprus and China while our A. ovis isolates clustered with those from Pakistan, China, and Sudan. In conclusion, this study reports the presence of Anaplasma spp. and A. ovis in Pakistani goats and recommends large-scale studies across diverse geo-climatic regions to further investigate the epidemiology, genetic diversity and host-parasite interactions for effective control of these infections in local goat populations.

Global warming significantly impacts coastal zones, particularly affecting ectothermic inhabitants such as bivalve mollusks. This study evaluates the response of the grooved carpet shell clam Ruditapes decussatus (Linnaeus, 1758) to increasing temperatures (22.5°C, 24.5°C, 26.5°C) over 25 days through the transcription of key genes involved in antioxidant defense [Cu‐Zn superoxide dismutase (Cu‐Zn sod), catalase, metallothionein], anti‐apoptotic procedures [b‐cell lymphoma 2 (bcl2)], and energy metabolism [pyruvate kinase (pk), phosphoenolpyruvate carboxykinase (pepck)]. Additionally, the genes catalase and Cu‐Zn sod were characterized for the first time, and along with the metallothionein gene, were sequenced in heat‐resilient and heat‐susceptible individuals to identify polymorphisms potentially associated with thermal tolerance. At 22.5°C, clams showed a delayed increase in glycolytic flux and a gradual up‐regulation of antioxidant and anti‐apoptotic mechanisms. At 24.5°C and 26.5°C, a strong initial stress response resulted in equally high mortality during the early days of exposure. Subsequently, clams appeared to shift toward a reduced energy metabolism, with mildly upregulated antioxidant defenses and anti‐apoptotic activity. With prolonged exposure, there was evidence of enhanced aerobic glycolysis, antioxidant, and anti‐apoptotic responses at these temperatures. However, increased pepck transcription on days 12 and 25 suggests that metabolic demands may have exceeded aerobic capacity, potentially triggering apoptotic processes at 26.5°C. Clams at 24.5°C maintained aerobic capacity upon the final day, also engaging anaerobic pathways to meet energy demands. Eventually, three SNPs were statistically correlated with heat resilience. These included one non‐synonymous SNP in catalase, one SNP in the 3′ untranslated region (3′UTR) of metallothionein, and one synonymous SNP in Cu‐Zn sod. These findings underscore the sensitivity of R. decussatus populations from the northeastern Mediterranean to persistent thermal stress and reveal several polymorphisms in antioxidant genes with potential adaptive significance. However, the limited sample size and the weak correlations observed in some cases highlight the need for further research to clarify the relationship between these polymorphisms and thermal resilience. Climate change is known to affect all levels of biology, setting at risk ecological balance. Among other organisms' groups, aquatic inhabitants, and particularly bivalves, are expected to be influenced to a greater extent owing to their inability to move to more mild temperatures. Therefore, these organisms represent excellent models to study the adaptation to global warming. Keeping this in mind, in the present study, we molecularly characterized the evolutionary response of the marine bivalve Ruditapes decussatus to increased seawater temperature by means of both antioxidant gene expression and resilience‐associated genetic loci. Several genes were characterized for the first time, and novel SNPs were detected that are linked with tolerance to climate change.

Ticks are the second most significant hematophagous ectoparasites, serving as vectors for numerous infectious diseases worldwide, surpassed only by mosquitoes. This study aimed to molecularly characterize ticks infesting cows and their associated pathogens in Khyber Pakhtunkhwa, Pakistan, with a focus on assessing the potential risk of zoonotic disease transmission. Between January 2023 and June 2024, 240 cows from six districts: Buner, Swat, Lower Dir, Upper Dir, Shangla, and Chitral, were examined for tick infestations. DNA was extracted using the phenol-chloroform method, and pathogen detection was performed via PCR targeting the gltA gene, while tick species were confirmed by amplifying partial 12 S rDNA sequences. The study revealed an average tick intensity of 12.73 ticks per infested cow and a mean abundance of 10.45 ticks per examined cow. In total, 2,507 ticks were collected and morphologically identified as Rhipicephalus microplus, comprising 915 females, 801 males, 510 nymphs, and 281 larvae. The highest tick burdens were observed in Buner and Swat, followed by Lower Dir, Upper Dir, and Shangla, with Chitral showing the lowest prevalence. Molecular screening detected multiple pathogens, notably the human pathogen Anaplasma capra and the cow pathogen Anaplasma marginale ; co-infections were occasionally observed. This paper presents the first phenotypic and phylogenetic characterization of A. capra in ticks from cows in Pakistan, emphasizing the zoonotic transmission risk. These findings advance our understanding of R. microplus associated pathogen risks and underscore the need for enhanced surveillance in the region.