Carlo Cabacang Lazado

Prospects of host-associated microorganisms in fish and penaeids as probiotics with immunomodulatory functions

Aquatic animals harbor a great number of microorganisms with interesting biological and biochemical diversity. Besides serving as the natural defense system of the host, the utilization potential of this microbial association has been identified particularly as reservoirs of candidate probiotics. Host-derived probiotics have gained popularity in recent years as they offer an alternative source of beneficial microbes to the industry that is customarily dependent on the use of terrestrial microorganisms. At present, there is an overwhelming number of candidate probiotics in aquaculture but their large-scale application is restricted by bio-technological concerns and fragmentary documented probiotic actions. This paper presents the current understanding on the use of probiotics as a sustainable alternative that promotes health and welfare in fish and penaeids. In particular, this paper discusses the relevance of host microbiota and its potential as a source of candidate probiotics. It also revisits the interaction between probiotics and host immunity to provide the foundation of the immunomodulatory functions of host-derived probiotics. Several studies demonstrating the immunomodulatory capabilities of host-derived candidate probiotics are given to establish the current knowledge and provide avenues for future research and development in this thematic area of probiotics research in aquaculture.

Probiotics as beneficial microbes in aquaculture: an update on their multiple modes of action: a review

ABSTRACT Wide and discriminate use of antibiotics has resulted in serious biological and ecological concerns, especially the emergence of antibiotic resistance. Probiotics, known as beneficial microbes, are being proposed as an effective and eco-friendly alternative to antibiotics. They were first applied in aquaculture species more than three decades ago, but considerable attention had been given only in the early 2000s. Probiotics are defined as live or dead, or even a component of the microorganisms that act under different modes of action in conferring beneficial effects to the host or to its environment. Several probiotics have been characterized and applied in fish and a number of them are of host origin. Unlike some disease control alternatives being adapted and proposed in aquaculture where actions are unilateral, the immense potential of probiotics lies on their multiple mechanisms in conferring benefits to the host fish and the rearing environment. The staggering number of probiotics papers in aquaculture highlights the multitude of advantages from these microorganisms and conspicuously position them in the dynamic search for health-promoting alternatives for cultured fish. This paper provides an update on the use of probiotics in finfish aquaculture, particularly focusing on their modes of action. It explores the contemporary understanding of their spatial and nutritional competitiveness, inhibitory metabolites, environmental modification capability, immunomodulatory potential and stress-alleviating mechanism. This timely update affirms the importance of probiotics in fostering sustainable approaches in aquaculture and provides avenues in furthering its research and development.

Host-derived probiotics Enterococcus casseliflavus improves resistance against Streptococcus iniae infection in rainbow trout (Oncorhynchus mykiss) via immunomodulation.

The present study evaluated the benefits of dietary administration of host-derived candidate probiotics Enterococcus casseliflavus in juvenile rainbow trout Oncorhynchus mykiss. Experimental diets were prepared by incorporating the microorganisms in the basal feed at 3 inclusion levels (i.e. 10(7) CFU g(-1) of feed [T1], 10(8) CFU g(-1) of feed [T2], 10(9) CFU g(-1) of feed [T3]). The probiotic feeds were administered for 8 weeks, with a group fed with the basal diet serving as control. The effects on growth performance, gut health, innate immunity and disease resistance were evaluated. Results showed that growth performance parameters were significantly improved in T2 and T3 groups. Activities of digestive enzymes such as trypsin and lipase were significantly higher in these two groups as well. Gut micro-ecology was influenced by probiotic feeding as shown by the significant increase in intestinal lactic acid bacteria and total viable aerobic counts in T2 and T3. Humoral immunity was impacted by dietary probiotics as total serum protein and albumin were significantly elevated in T3. The levels of serum IgM significantly increased in all probiotic fed groups at week 8; with the T3 group registering the highest increment. Respiratory burst activity of blood leukocytes were significantly improved in T2 and T3. Hematological profiling further revealed that neutrophil counts significantly increased in all probiotic fed groups. Challenge test showed that probiotic feeding significantly improved host resistance to Streptococcus iniae infection, specifically in T2 and T3 where a considerable modulation of immune responses was observed. Taken together, this study demonstrated E. casseliflavus as a potential probiotics for rainbow trout with the capability of improving growth performance and enhancing disease resistance by immunomodulation.

Dietary phytoimmunostimulant Persian hogweed (Heracleum persicum) has more remarkable impacts on skin mucus than on serum in common carp (Cyprinus carpio)

Immunostimulation through the use of sustainable and eco-friendly dietary additives is one of the current prophylactic strategies in fish husbandry. Plant-based immunostimulants are highly considered for this intent, both for their scientific and practical advantages. Persian hogweed (Heracleum persicum) is a flower-bearing herb that possesses interesting pharmacological importance due to its bioactive compounds. It is commonly used as spice, food additive, dietary supplement and traditional remedy. The present study evaluated the potential of H.xa0persicum as a dietary phytoimmunostimulant in common carp (Cyprinus carpio). The powder form of H.xa0persicum was incorporated in the basal diet at three different inclusion levels: 2.5, 5 and 10xa0gxa0kg-1. The basal diet (0xa0gxa0kg-1 of H.xa0persicum) served as control. Experimental diets were administered to the fish for a period of 8 weeks. At the termination of the feeding experiment, impacts on fish immunity and performance were evaluated. Inclusion of H.xa0persicum in the diet significantly elevated several immunological factors such as immunoglobulins, lysozyme, protease and alternative complement activities in carp. Interestingly, the changes were more pronounced in the skin mucus than in the serum. Performance was significantly improved in the fish groups that received the candidate phytoimmunostimulant. Specifically, final weight, weight gain, specific growth rate and feed conversion ratio were significantly improved in the fish that received dietary H.xa0persicum at inclusion levels 5xa0gxa0kg-1 and higher. This study demonstrated the potential of Persian hogweed as a candidate dietary phytoimmunostimulant in carp, impacting mainly the skin mucosal defenses. The study supports the current trend in the exploration of sustainable plant-based dietary supplements that are capable of boosting the immunological defenses of farmed fish.

Humoral and mucosal defense molecules rhythmically oscillate during a light-dark cycle in permit, Trachinotus falcatus.

Circadian rhythm provides organisms with an internal system to maintain temporal order in a dynamic environment. This is typified by a 24-h cycle for a number of physiological processes, including immunity. The present study characterized the humoral and mucosal defense molecules and their dynamics during a light-dark (LD) cycle in juvenile permit, Trachinotus falcatus. All studied defense molecules were constitutively identified in serum and skin mucus. Serum generally exhibited higher levels of these defenses than skin mucus, with the exception of anti-protease (ANTIPRO). The difference in ANTIPRO, lysozyme (LYZ), esterase (ESA) and catalase (CAT) levels between serum and skin mucus was not affected by the phase of the daily cycle. However, a clear phase-dependent difference was observed in protease (PRO), globulin (GLOB), myeloperoxidase (MPO), alkaline phosphatase (ALP) and glutathione peroxidase (GPX) levels. Activities of ALP and GPX displayed significant daily rhythmicity in both serum and skin mucus. Circadian profile of ALP was identical in both biofluids, but an antiphasic feature was exhibited by GPX. GLOB and MPO levels also exhibited significant daily oscillation but only in serum with acrophases registered at ZT 14.5 and 6.15, respectively. Mucus PRO and serum ANTIPRO demonstrated significant temporal variations during a daily cycle albeit not rhythmic. Cluster analysis of the defense molecules in serum and skin mucus revealed two different daily profiles suggesting a possibility of distinct circadian control between humoral and mucosal immunity. These observations indicate that LD cycle had a remarkable impact in the defense molecules characterizing the humoral and mucosal immunity in permit. Daily rhythmic patterns of these defense molecules contribute to our understanding of the barely explored interplay of immunity and circadian rhythm in teleost fish. Lastly, the results could be useful in developing aquaculture practices aiming at modifying the immune functions of permit for improved health.

Innate immune defenses exhibit circadian rhythmicity and differential temporal sensitivity to a bacterial endotoxin in Nile tilapia (Oreochromis niloticus)

The present study investigated the daily dynamics of humoral immune defenses and the temporal influence in the sensitivity of these responses to a bacterial endotoxin in Nile tilapia (Oreochromis niloticus). The first experiment subjected the fish to two photoperiod conditions, 12L:12D (LD) and 0L:24D (DD), for 20 days to characterize the rhythms of humoral immunity. Serum alkaline phosphatase (ALP), lysozyme (LYZ), peroxidase (PER) and protease (PRO) exhibited significant rhythmicity under LD but not in DD. No significant rhythms were observed in esterase (ESA) and anti-protease (ANTI) in both photoperiod conditions. Fish reared under LD were subsequently subjected to DD while the group previously under DD was exposed to LD, and this carried on for 3 days before another set of samples was collected. Results revealed that the rhythms of LYZ, PER and PRO but not ALP persisted when photoperiod was changed from LD to DD. Nonetheless, immune parameters remained arrhythmic in the group subjected from DD to LD. Cluster analysis of the humoral immune responses under various light conditions revealed that each photic environment had distinct daily immunological profile. In the second experiment, fish were injected with bacterial endotoxin lipopolysaccharide (LPS) either at ZT3 (day) or at ZT15 (night) to evaluate the temporal sensitivity of humoral immunity to a pathogen-associated molecular pattern. The results demonstrated that responses to LPS were gated by the time of day. LPS significantly modulated serum ALP and ANTI activities but only when the endotoxin was administered at ZT3. Serum LYZ and PER were stimulated at both injection times but with differing response profiles. Modulated LYZ activity was persistent when injected at ZT3 but transient when LPS was applied at ZT15. The magnitude of LPS-induced PER activity was higher when the endotoxin was delivered at ZT3 versus ZT15. It was further shown that plasma cortisol was significantly elevated but only when LPS was administered at ZT3. On the other hand, plasma melatonin was significantly affected by LPS injection but only when exposed at ZT15. Taken together, this study shows that several key components of humoral immunity in tilapia exhibit circadian rhythms and adapt to photoperiodic changes. Further, results of the bacterial endotoxin challenge suggest that responsiveness of serum humoral factors to a biological insult is likely mediated by the time of day, highlighting the importance of circadian rhythm in the immunological functions of fish.

Interplay between daily rhythmic serum-mediated bacterial killing activity and immune defence factors in rainbow trout (Oncorhynchus mykiss)

ABSTRACT Circadian rhythm is emerging as an important regulator of immune functions. However, there is a paucity of information on the influence of this biological phenomenon in the antimicrobial factors in teleost fish. This study investigated the dynamics and interplay of serum‐mediated bacterial killing activity and immune defence factors throughout the light:dark (LD) cycle in rainbow trout (Oncorhynchus mykiss). The juvenile fish came from two different emergence time fractions (i.e., late and early) that were believed to exhibit behavioural and physiological differences. Serum collected during the day from fish (mean ± SD: 39.8 ± 6.3 g) reared under 14L:10D photoperiod demonstrated bactericidal activity against Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida of varying magnitude, but no significant differences between the emergence fractions were observed. A day‐night comparison in the same batch of fish revealed time‐of‐day dependence in the bactericidal activity against F. psychrophilum and Y. ruckeri amongst emergence fractions. A group of fish (63.3 ± 4.7 g) from each fraction was entrained to 12L:12D photoperiod for 21 days to investigate whether serum bactericidal activity exhibited daily rhythm. Serum‐mediated bacterial killing activity against F. psychrophilum and Y. ruckeri displayed significant daily rhythm in both emergence fractions, where the peak of activity was identified during the light phase. Moreover, several serum defence factors manifested variations during the LD cycle, where anti‐protease (ANTI) and myeloperoxidase (MPO) activities exhibited significant daily oscillation. However, there were no remarkable differences in the daily changes of serum factors amongst emergence fractions. Acrophase analysis revealed that the peaks of activity of alkaline phosphatase (only in late fraction), ANTI, lysozyme (only in early fraction) and MPO were identified during the light phase and corresponded with the period when serum‐mediated bacterial killing activity was also at its highest. The daily dynamics of bactericidal activity and immune defence factors displayed positive correlation, particularly between MPO and, the two pathogens (i.e., F. pyschrophilum and Y. ruckeri). Taken together, the study revealed that serum‐mediated bacterial killing activity and immune defence factors remarkably varied during the LD cycle in rainbow trout. In addition, the two emergence fractions displayed nearly comparable immunological profiles.

Short-term feed and light deprivation reduces voluntary activity but improves swimming performance in rainbow trout Oncorhynchus mykiss

Rainbow trout Oncorhynchus mykiss (~u2009180xa0g, 16xa0°C and <u20095xa0kgxa0m−3) that were feed deprived and kept in total darkness showed a significant increase in critical swimming speed (Ucrit) between 1 and 12xa0days of deprivation (from 3.35 to 4.46 body length (BL)xa0s−1) with no increase in maximum metabolic rate (MMR). They also showed a significant decrease in the estimated metabolic rate at 0 BLxa0s−1 over 12xa0days which leads to a higher factorial aerobic metabolic scope at day 12 (9.38) compared to day 1 (6.54). Routine metabolic rates were also measured in ~u200990xa0g rainbow trout that were swimming freely in large circular respirometers at 16xa0°C. These showed decreasing consumption oxygen rates and reductions in the amount of oxygen consumed above standard metabolic rate (a proxy for spontaneous activity) over 12xa0days, though this happened significantly faster when they were kept in total darkness when compared to a 12:12-h light–dark (LD) photoperiod. Weight loss during this period was also significantly reduced in total darkness (3.33% compared to 4.98% total body weight over 12xa0days). Immunological assays did not reveal any consistent up- or downregulation of antipathogenic and antioxidant enzymes in the serum or skin mucus of rainbow trout between 1 and 12xa0days of feed and light deprivation. Overall, short periods of deprivation do not appear to significantly affect the performance of rainbow trout which appear to employ a behavioural energy-sparing strategy, albeit more so in darkness than under a 12:12-h LD regime.

Rhythmicity and plasticity of digestive physiology in a euryhaline teleost fish, permit (Trachinotus falcatus)

Digestive physiology is considered to be under circadian control, but there is little evidence in teleost fish. The present study explored the rhythmicity and plasticity to feeding schedules of enzymatic digestion in a candidate aquaculture fish, the permit (Trachinotus falcatus). The first experiment identified the rhythms of digestive factors throughout the light-dark (LD) cycle. Gastric luminal pH and pepsin activity showed significant daily variation albeit not rhythmic. These dynamic changes were likewise observed in several digestive enzymes, in which the activities of intestinal protease, chymotrypsin and lipase exhibited significant daily rhythms. In the second experiment, the existence of feed anticipatory activity in the digestive factors was investigated by subjecting the fish to either periodic or random feeding. Anticipatory gastric acidification prior to feeding was identified in periodically fed fish. However, pepsin activity did not exhibit such anticipation but a substantial postprandial increase was observed. Intestinal protease, leucine aminopeptidase and lipase anticipated periodic mealtime with elevated enzymatic activities. Plasma melatonin and cortisol demonstrated robust daily rhythms but feeding time manipulations revealed no significant impact. Plasma ghrelin level remained constant during the LD cycle and appeared to be unaffected by differing feeding regimes as well. Taken together, the digestive factors of permit were highly dynamic during the LD cycle. Periodic feeding entrained digestive physiology and mediated anticipatory gastric acidification and intestinal enzymatic activities. This knowledge will be essential in developing feeding protocols and husbandry-related welfare strategies that will further advance this candidate finfish as an aquaculture species.

Cortisol levels and expression of selected stress and apoptosis-related genes in the embryos of Atlantic cod, Gadus morhua following short-term exposure to air

Embryos (morula stage) of Atlantic cod, Gadus morhua L., were collected and subjected to air exposure for 2xa0min. followed by recovery at ambient conditions in the rearing container. Total immunoreactive cortisol and transcription of selected stress- and apoptosis-related genes of the embryos were determined before the application of the stressor and at 0.5, 1 and 24xa0h post-exposure. There was no significant difference in the total cortisol levels of the fertilized eggs before and after handling stress. There was high expression level of hsp70 and sod before application of the stressor and significantly increased at 0.5xa0h post-exposure. The expression levels of cat and gpx were weak to moderate and were not affected by the stressor. The apoptotic genes, mcl1 and NR-13 were highly expressed and significantly increased after exposure to air. Bcl-X1 and Bcl-X2 were moderately expressed in the control samples, but only the expression level of Bcl-X1 significantly increased following exposure to air. Cluster analysis of the different gene expression levels indicated three categories: those genes that did not show any change in the expression levels post-air exposure; those that had low expression level in the control followed by a significant increase after air exposure; and those that had high expression levels in the control followed by a further increase in expression after air exposure. These results clearly demonstrate that there are potential molecular biomarkers of the response in cod embryos as a consequence of air exposure at a time when cortisol is not fully active.