Combined Pituitary Hormone Deficiency in lhx4-Knockout Zebrafish.

LIM homeobox 4 (LHX4) is a transcription factor crucial for anterior pituitary (AP) development. Patients with LHX4 mutation suffer from combined pituitary hormone deficiency (CPHD), short statures, reproductive and metabolic disorders and lethality in some cases. Lhx4-knockout (KO) mice fail to develop a normal AP and die shortly after birth. Here, we characterize a zebrafish lhx4-KO model to further investigate the importance of LHX4 in pituitary gland development and regulation. At the embryonic and larval stages, these fish express lower levels of tshb mRNA compared with their wildtype siblings. In adult lhx4-KO fish, the expressions of pituitary hormone-encoding transcripts, including growth hormone (gh), thyroid stimulating hormone (tshb), proopiomelanocortin (pomca) and follicle stimulating hormone (fshb), are reduced, the pomca promoter-driven expression in corticotrophs is dampened and luteinizing hormone (lhb)-producing gonadotrophs are severely depleted. In contrast to Lhx4-KO mice, Lhx4-deficient fish survive to adulthood, but with a reduced body size. Importantly, lhx4-KO males reach sexual maturity and are reproductively competent, whereas the females remain infertile with undeveloped ovaries. These phenotypes, which are reminiscent of those observed in CPHD patients, along with the advantages of the zebrafish for developmental genetics research, make this lhx4-KO fish an ideal vertebrate model to study the outcomes of LHX4 mutation.

Involvement of inducible nitric oxide synthase (iNOS) in immune-functioning of Paphia malabarica (Chemnitz, 1782).

In recent years, the role of inducible nitric oxide synthase (iNOS) isoform has been widely studied because of its immunological relevance in higher organisms as well as invertebrates including bivalves. However, little is known about the immunological role of iNOS in Paphia malabarica defense mechanism. In this study, we immunodetected the presence of iNOS in P. malabarica hemocytes using antibody N9657 monoclonal anti-nitric oxide synthase. In addition, increased iNOS activity was evident in response to a higher bacterial dosage (Vibrio parahaemolyticus and V. cholerae), highlighting the dose-dependent iNOS activity induction. Also, higher bacterial survivability was observed in the presence of iNOS inhibitor, i.e., S-methylisothiourea hemisulphate (SMIS) thus, validating the bactericidal role of iNOS. These findings implicate the involvement of iNOS in immune-functioning of P. malabarica. Future work should focus on elucidating the expression and regulation of pathogenesis in P. malabarica, involving iNOS.

Immunoecology of the short neck clam Paphia malabarica (Chemnitz, 1782) in a tropical monsoon-influenced estuary.

Understanding the variability in organism's immunological response is crucial for predicting changes at population or community level. The present study investigated the immunoecology of a commercially valuable clam Paphia malabarica in a tropical monsoon-influenced estuary. Clams were collected monthly during a year cycle, which coincided with pre-monsoon (February-May), monsoon (June-September) and post-monsoon seasons (October-January). For assessment of immune functioning, selected hemocyte parameters (total hemocyte concentration, hemocyte mortality, lysosomal content, esterase activity, reactive oxygen species production, and phagocytic activity) were analyzed using flow cytometry. Simultaneously, clam's condition index, nutrients, chlorophyll a, dissolved oxygen, pH, temperature and bacterial density were also measured at the sampling site. Our results exhibited seasonal patterns in hemocyte functioning with the highest activity during the pre-monsoon season (suggestive of a suitable harvesting period) and lowest during monsoon (suggestive of a critical biological period). The critical biological period for P. malabarica was marked with compromised immune parameters inflicted by low salinity, food availability, and possibly high bacterial abundance. Also, the involvement of reproductive stress altering the hematological functioning in P. malabarica cannot be ruled out. Nutrients, dissolved oxygen, pH and temperature could not explain much of the hemocyte variability. The present study has further validated the usefulness of hemocyte as a suitable marker for understanding immunoecology of P. malabarica which is of prime importance, especially in a monsoon-influenced tropical estuarine environment. The findings of our research will be constructive in monitoring natural as well as cultivated bivalve populations of economic and ecological relevance.

Effect of pea crab Pinnotheres vicajii (Chhapgar, 1957) on immunocompetence of bivalve Paphia malabarica (Chemnitz, 1782).

The endosymbiont-host relationship between a pea crab and its bivalve host has been studied previously using bivalve's multi-physiological parameters. The present study is first of its kind that uses hemocyte's immune functionality to evaluate the symbiotic relationship between bivalve Paphia malabarica and its symbiont crab, Pinnotheres vicajii. The sampling was carried out at two stations (differed in nutrient concentration, productivity, and bacterial abundance) located along the southwest coast of India. The results showed lower immunocompetence in the case of bivalves infested with pea crabs. The lowering of hemocyte population was the most prominent and perhaps the initial reaction in response to focal inflammation or wound inflicted by the pea crabs. A significant lowering of phagocytosis, lysozyme, and esterase activity was also observed, indicating the detrimental impact of crab infestation. Interestingly, no significant correlation was found between parasitism and condition index despite the lower immunocompetence, validating the effectiveness of using hematological parameters in comparison to other life-history traits. This indicates that the immune parameters being directly related to the organism's metabolic state can elucidate the early effect of stressors, therefore, proving to be a better proxy for understanding a host-pathogen relationship. Disparity found in bivalve's immunocompetence between the sampling sites could be linked to either variation in environmental parameters, the size of the symbiont or the infection level. Our preliminary results provide a direction towards examining immunodepression in parasitized bivalves by considering ex-situ controlled parasitism induction along with varying environmental conditions. In view of this, efforts must be directed towards minimizing pea crab's outbreaks, especially in cultivated shellfish farms. Future studies should elucidate molecular mechanisms involved in the immunocompetence of P. malabarica, hosting the parasitic pea crabs.

Immune response of the short neck clam Paphia malabarica to salinity stress using flow cytometry.

Paphia malabarica is a predominant and commercially important bivalve in India, persistently challenged by wavering salinity in a monsoon-influenced estuary. To examine the organism's immunological response under such a condition we challenged P. malabarica with different salinities (0, 5, 15, 25 and 35) for varied periods using a two-way experimental approach (in vitro and in vivo). This is the first study to report the response of P. malabarica hemocytes to salinity stress from a monsoon-influenced estuary on the southwest coast of India. Evaluation of total hemocytes count, mortality, lysosomal content, reactive oxygen species production, phagocytic and esterase activity was carried out using flow cytometric analysis. In both the experimental conditions, hemocyte parameters were significantly compromised at lower salinities (0 and 5) with an evident immuno-salinity tolerance range of 15-35. The damaging impact of 0 and 5 salinities on hemocyte function intensified with a longer exposure period, indicating that prolonged exposure to low salinity could be detrimental to bivalve wellness if they are pushed beyond their tolerance range which is usually observed during the monsoon. Further studies should focus on the interactive effect of salinity tagged with different stressors influencing biology of P. malabarica.