Transcriptomic Variation of Amphiprion Percula (Lacepède, 1802) in Response to Infection with Cryptocaryon Irritans Brown, 1951.

Cryptocaryon irritans (Brown 1951) frequently infect the Pomacentridae fishes causing severe economic losses. However, the anti-C. irritans' molecular mechanism in these fishes remains largely unknown. To address this issue, we conducted RNA-Seq for C. irrtians-infected gills of the clownfish Amphiprion percula (Lacepède 1802) at the early (day 1) and late (day 3) stages of infection. A total of 1655 differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs showed a vast genetic variation related to the following aspects: ECM-receptor interaction, P13K-Akt signalling, cytokine-cytokine receptor interaction, and endocytosis. During the early phase of infection, key genes involved in ATP production, energy homeostasis, and stress control were abruptly increased. In the late phase, however, acute response molecules of the peripheral nervous system (synaptic transmission and local immunity), metabolic system triggering glycogen synthesis, energy maintenance, and osmoregulation were found to be critical. The highest number of upregulated genes (URGs) recovered during the early phase was included under the 'biological process' category, which primarily functions as response to stimuli, signalling, and biological regulation. In the late phase, most of the URGs were related to gene regulation and immune system processes under 'molecular function' category. The immune-related URGs of early infection include major histocompatibility complex (MHC) class-II molecules apparently triggering CD4+ T-cell-activated Th responses, and that of late infection include MHC class-1 molecules for the possible culmination of CD8+ T-cell triggered cytotoxicity. The high level of genic single nucleotide polymorphisms (SNPs) identified during the late phase of infection is likely to influence their susceptibility to secondary infection. In summary, the identified DEGs and their related metabolic and immune-related pathways and the SNPs may provide new insights into coordinating the immunological events and improving resistance in Pomacentridae fishes against C. irritans.

Modern biotechnological strategies for vaccine development in aquaculture - Prospects and challenges.

Advances in genomics and the gradual reduction of cost for technologies like whole-genome sequencing have provided exciting opportunities for developing modern biotechnological-based vaccines in aquaculture. This systemic review describes the prospects and challenges of implementing these high-tech vaccines in fish species. The majority of the commercial vaccines in aquaculture utilize conventional procedures for which cost of administration, protective immunity and safety issues are the major challenges. In recent years, more efficient vaccines are being developed by adopting the advances in vaccine technology. Vaccines based on surface antigens, protein/peptide/polysaccharide subunits, recombinant DNA/mRNA/plasmids, novel antigen expression and delivery systems (bacteriophage particles, virus like particles/VLPs, recombinant yeast, mucosal vaccines), novel molecular adjuvants (IL-8, IL-12, HSPs), and encapsulation polymers and polysaccharides like chitosan nanoparticles and PLGA microcapsule were successfully developed. These biotechnology-based vaccines have proved to be very efficient in field trials, but are always in the research pipeline or as patents. Only very few of them are licensed for use, that too, in high-valued fishes like salmonids. Currently, commercial aquaculture vaccines are available for Aeromonas salmonicida, Vibrio salmonicida, Yersinia ruckeri, Vibrio anguillarum, Edwardsiella ictalurid, and for certain Betanodaviruses. Nevertheless, no registered vaccines are available for other major infectious diseases/pathogens such as viral hemorrhagic septicemia virus (VHSV), viral nervous necrosis virus (VNN) and certain other betanodaviruses, channel catfish virus (CCV), gill disease bacteria, mycobacteria, flavobacterium, Edwardsiella tarda, and certain streptococci. Despite the important economic losses that the pathogens cause to aquaculture worldwide, the commercialization of vaccines remains limited due to immunological pitfalls in aquatic species, large-scale vaccination issues, unregulated use of antibiotics and chemicals, gene-based vaccine regulations and commercial viability. If attempts are to be made to develop novel delivery methods, cost-effective procedures, and relaxations in DNA vaccine regulations, biotechnology-based vaccination could circumvent the emerging disease challenges in aquaculture.

Immune responses of Cyprinus carpio induced by protein extracts of Lernaea cyprinacea Linnaeus, 1758.

Lernaea cyprinacea Linnaeus, 1758 is an ectoparasite showing widespread infections in tropical aquaculture, and the present study aimed to determine the specific immune responses against this parasite. For the experiment, whole parasite extracts were injected intraperitoneally into Cyprinus carpio Linnaeus, 1758, and samples of epidermal mucus and blood were drawn at 0, 1, 7 and 14-days post-injection (DPI). The results revealed high levels of protein, protease and lysozyme activities in the experimental fish which were injected with L. cyprinacea protein extract. In the epidermal mucus, the total protein concentration of the control fish was 460 µg/mL, and the level raised significantly to 800 µg/mL in the experimental fish. The lysozyme activity increased from 741.5 u/mL to a peak level of 1448.5 u/mL at 7DPI. The protease activity was also found elated gradually from 2.91 u/µL to 4.49 u/µL at 1 to 14 DPI. In the serum samples, the protein concentration remained steady throughout the experiment period. However, all the experimental fish displayed statistically high levels of lysozyme and protease activity, from 890 u/mL to 1220 u/mL, and 6.10 u/µL to 11.88 u/µL, respectively. In the whole blood samples, the haemoglobin content and the red blood cells (RBC) count did not show any significant change in any of the experimental groups. But, the percentage of lymphocytes showed a marginal increase from 0.47 to 0.6 in the experimental groups. Overall, the immune responses induced by L. cyprinacea protein extracts depicts a pattern of specific responses, in which the local humoral responses dominate the systemic humoral/cellular response. The results further revealed the possibility of futuristic approaches to control freshwater ectoparasites.

The occurrence of parasitic copepods and isopods infesting the marine teleost fishes of Kerala coast, India.

The present study reports the occurrence of parasitic copepods and isopods infecting marine teleost fishes from the Kerala coast, India. A total of 1795 fishes belonging to 38 species were collected from the fish landing centres across the Kerala Coast for 5 months. The isopod & copepod infection status of these fishes were assessed. The incidents of site-specific infection were documented for all parasites and a fecundity analysis was conducted in randomly selected species. The single or multiple parasitic crustacean infestation and host preference (single or multiple hosts) were also documented. A total of 32 species of copepods and 6 species of isopod were sourced out. However, the maximum prevalence was recorded for the family Lernanthropidae (71.43%) and the maximum intensity (14) was recorded for a Caligid copepod Euryphorus nordmannii. The predominantly targeted region of infection on the host fish appeared to be gill filament (52.15%). The fecundity showed significant differences between the tested species. Fourteen species of fishes showed multiple parasitic crustacean infestation and eight species of parasites infected multiple hosts. The present study demonstrates that over 68% of marine fish species of Kerala coast were under heavy infection either by isopods or copepods.

Protandrous Hermaphroditic Reproductive System in the Adult Phases of Mothocya renardi (Bleeker, 1857) (Cymothoidae: Isopoda: Crustacea) -Light and Electron Microscopy Study.

The reproductive system of Mothocya renardi (Bleeker, 1857), a protandrous hermaphroditic cymothoid that infects the belonid fish Strongylura leiura Bleeker, 1850, is characterized using light and electron microscopy. Three protandrous hermaphroditic adult phases are identified: male, transitional and female. Each phase includes a paired reproductive system, one on either side of the gut. Each consists of three lobed testes, followed by an ovary, then a vas deferens that opens into a penis on the same side. During the male phase, all testis lobes are filled with germ cells at various stages of spermatogenesis and spermeogenesis. Primary and secondary spermatogonial cells are confined to the peripheral side of the testis lobe. The ovary shows peripheral germarium and a large number of yolkless oocytes encircled by follicle cells. The oviduct emerged from the ovary mid laterally and its distal end was found to be sealed. The exceptionally elongated spermatozoon consists of a head and a long filamentous tail. The spermatozoa are found organized into characteristic bundles to form spermatophores, and these are also packed in the vas deferens during the male phase. During the transitional phase, on the other hand, testes appear to be withered, but the vas deferens contains spermatophores. The ovary shows yolky oocytes encircled by follicle cells. During the female phase, the testis lobes appear as thin, empty, and sac like, and the extremity of the vas deferens is closed. Ovaries contain yolky oocytes and more prominent oviducts than male and transitional phases. The present paper also discusses the pattern of correlation between 1) the ovarian and brood cycles and 2) the ovarian and molt cycles.

Reproductive System in the Male Phase of a Parasitic Isopod (Crustacea) - Morphological, Histological and Ultrastructural Evidence for Sequential Protandrous Hermaphroditic Changes.

This paper reports the protandric hermaphroditic changes in the reproductive system of the male-phased Norileca indica, a cymothoid that parasitizes the scombrid fish Rastrelliger kanagurta. Each part of N. indica's paired reproductive system lies on either side of the gut. This study considers the three successive size classes of the male phase - designated as M1, M2 and M3 - using light microscopy and ultrastructural methods. The testis comprises of three bulged sac-like lobes labelled t1, t2 and t3, all of which open into the ovary of their respective side. The vas deferens, which emerges as a posterior extension of the ovary, opens into the penis and the distal end of each oviduct leads to a sealed gonopore on their respective sides. Each testis lobe (t1/t2/t3) displays clusters of germ cells undergoing stage-specific differentiation. Spermatids undergoing sequential changes associated with spermiogenesis keep close proximity to somatic accessory cells. The characteristic histological changes associated with protandric hermaphroditism are visible in the ovaries of sequential size classes (M1, M2 and M3). In early M1, besides spermatophores, the ovary has abundant polymorphic nuclei; in the mid/late M1, the posterior ovary has abundant spermatophores, anterior displayed oogonia, previtellogenic oocytes and two distinct forms of follicle cells. In M2, the anterior ovary shows compactly arranged oocytes while the posterior region accommodates spermatophores - fewer, however, than during M1. The entire ovary during M3 is crowded with previtellogenic oocytes, which marginalize the spermatophore passage. The vas deferens of the smallest M1 lack spermatophores. As the size class progresses through late M1 into M2 and M3, the posterior vas deferens is filled with spermatophores, which closely associate with the glandular epithelial lining.

Norileca indica (Crustacea: Isopoda, Cymothoidae) Infects Rastrelliger kanagurta Along the Malabar Coast of India - Seasonal Variation in the Prevalence and Aspects of Host-parasite Interactions.

This paper reports seasonal variations in the prevalence of host-parasite associations between Norileca indica, a cymothoid, and the Indian mackerel Rastrelliger kanagurta along the Malabar Coast of India. Eighty-eight marine fish species belonging to diverse families were examined, and only R. kanagurta was shown to be parasitized by N. indica, indicating a narrow host specificity. The prevalence, mean intensity, and abundance were 30.70, 1.71, and 0.52%, respectively. Different life cycle of this parasite, from larva to adult, infect the host fish. In most instances, a pair of N. indica infected the host, and in these instances a male-female combination was the most common (89.17%). The monthly occurrence of N. indica was charted for a period of 38 months (July 2012 to July 2014; March 2017 to March 2018), and statistical comparison of the data showed a significant difference (p < 0.001) among seasons. A positive correlation (r = 0.40) was observed between the size of female parasites and that of their respective host fish. There was a positive correlation (r = 0.78) between the size of female parasites and their fecundity. In all instances, adult N. indica individuals were found to specifically attach to the mucus membrane of branchial operculum, causing visible physical damage, including atrophy (reduced length) of the gill filaments and overall loss of gill normalcy. Furthermore, permanent occupancy by female N. indica resulted in the formation of a deep pit in the gill chamber floor and also caused atrophy of gill filaments. Overall, our findings yielded a greater understanding of the occurrence, season-wise prevalence, and potential host- parasite associations of N. indica.

Branchial cymothoids infesting the marine food fishes of Malabar coast.

Occurrence of cymothoid isopods parasitizing the branchial chamber of marine food fishes along the Malabar coast was investigated. Live and fresh fishes collected from the Ayyikkara fish landing center (Lat. 11°51'N, Long. 75°22'E; Malabar coast, India) were subjected to the thorough observation for the presence of branchial cymothoids for 3 consecutive years (November 2009-November 2012). Among the recovered cymothoids, 11 species were branchial residents belonging to 6 genera; the species include Agarna malayi, Catoessa gruneri, C. boscii, Joryma hilsae, J. brachysoma, J. engraulidis, J. sawayah, Mothocya collettei, M. renardi, Norileca indica and Ryukyua circularis; highest prevalence being exhibited by two species of Mothocya, (M. renardi and M. collettei) parasitizing the belonidaen fishes, Strongylura leiura (92.15 %) and Tylosurus crocodilus crocodilus (87.2 %) respectively. Except Mothocya species, which preferred the branchial floor for infestation, all recovered branchial cymothoids were found attached the inner wall of the operculum. In several instances, the parasites appeared in male-female pairs, one in each branchial cavity. Ovigerous female members of all species of branchial cymothoids except R. circularis showed remarkable bending either towards left or right depending on whether they are located in right or left branchial cavity of their respective host fishes. The deleterious effects of parasitization by all recovered branchial cymothoids include the formation of a pit like depression in the branchial chamber and atrophy of the gill filament; the damage was more pronounced in the gill cavity of parasitized host fishes where the ovigerous female member was accommodated.

Mothocya renardi (Bleeker, 1857) (Crustacea: Isopoda: Cymothoidae) parasitising Strongylura leiura (Bleeker) (Belonidae) off the Malabar coast of India: Redescription, occurrence and life-cycle.

Mothocya renardi (Bleeker, 1857), a protandrically hermaphroditic cymothoid, parasitising the banded needle fish Strongylura leiura (Bleeker) from the Malabar Coast, India is redescribed and morphological data for different life-cycle stages [male, transitional and ovigerous female, larvae (pre-manca and manca) and juvenile] are provided. Mothocya renardi exhibited strict oligoxenous host specificity by infesting only S. leiura and showed high prevalence levels (reaching up to 92%). The life-cycle of M. renardi comprises three major phases (marsupial phase, free living phase and infestive phase). The marsupial phase comprised one zygotic, three embryonic and two larval stages, all of which remained in the marsupium until the final staged manca is released into the surrounding water. After having led a short free- swimming life, the manca infested the branchial cavity of the host fish, S. leiura. Subsequently it was transformed successively into juvenile, male, transitional and finally functional female through biphasic moult which occurs in between each stage. Based on the presence (or absence) of a brood pouch and/or marsupiumites, six successive stages of the female population were also identified. These data will help precise identification of the female M. renardi irrespective of their stage. The present paper also discusses the host-parasite interactions between S. leiura and M. renardi.

Cymothoa frontalis, a cymothoid isopod parasitizing the belonid fish Strongylura strongylura from the Malabar Coast (Kerala, India): redescription, description, prevalence and life cycle.

BACKGROUND: Cymothoa frontalis Milne Edward, 1840 is a very poorly described cymothoid, notwithstanding the previous redescription of the female. Pertinently, to date, the host of C. frontalis has not been identified with adequate precision. Most of the descriptions of cymothoids carried out hitherto were based primarily on females, but practically ignoring other life cycle stages. The present paper redescribes the female and describes other life cycle stages of the species C.frontalis toget better precision in their identification. RESULTS: The female phase of C. frontalis is redescribed according to type specimens extant in the NMNH, Paris, and also by the data obtained from live specimens collected during the present study. The general morphology and appendages of various life cycle stages of the species are described. Among 80 fish species from 35 families examined, C.frontalis wasrecovered only from Strongylurastrongylura signifyingits oligoxenous host specificity, the prevalence and intensity being 68.65% and 1.9, respectively. Each host fish in more than 85% of the population was infested with a pair of C. frontalis, in three combinations, predominantly with male-female pair (70.9%). C. frontalis exhibitedstrict site specificity attaching to the buccal cavity of the host fish. The study has also identified three major phases (marsupial, free living and infective) in the life cycle of C. frontalis. The zygotic-staged marsupiumites were developed through five sequential ontogenetic stages. The manca released from the marsupium become infective aftera brief period of free swimming life. During the infective phase, C.frontalis completesremaining life cycle stages with successive moulting. Further, six successive stages of the ovigerous females have also been identified. CONCLUSIONS: Thepresent redescription of the female and the description of transitional, male, juvenile and larvae of C. frontalis facilitate precise identification of the species at any stage of the life cycle. Further, the strict host and site specificitiesof the parasite, as borne out from the present study, and its high degree of prevalence in the host make C. frontalis as an ideal model organism to study the strategies to be adopted for the management of parasites infesting edible fishes.

Alternative splicing in the fiddler crab cognate ecdysteroid receptor: variation in receptor isoform expression and DNA binding properties in response to hormone.

RXR cDNA cloning from three Uca species led to the identification of 4 conserved isoforms, indicative of alternative splicing in the hinge and ligand binding domains (LBD). Sequencing of overlapping clones from a Ucapugilator genomic library identified EcR isoforms matching previously identified cDNA variants; in addition, a cryptic exon in the LBD was detected and evidence for expression of this new isoform was obtained from next-generation sequencing. RNA-seq analysis also identified a new amino terminal EcR variant. EcR and RXR transcript abundance increases throughout ovarian maturation in U. pugilator, while cognate receptor transcript abundance remains constant in a related Indo-Pacific species with a different reproductive strategy. To examine if crab RXR LBD isoforms have different physical properties in vitro, electromobility shift assays were performed with different EcR isoforms. The cognate crab and fruit fly receptors differ in their responses to hormone. Ecdysteroids did not increase DNA binding for the crab heterodimers, while ecdysteroids stimulate binding for Drosophilamelanogaster EcR/USP heterodimers. In swapping experiments, UpEcR/USP heterodimers did not show ligand-responsive differences in DNA binding; both crab RXR LBD isoforms, however, conferred ligand-responsive increases in DNA binding with DmEcRs. These data indicate that both UpRXR LBD isoforms can heterodimerize with the heterologous DmEcR receptors and promote ligand and DNA binding. Unresponsiveness of the cognate receptors to ecdysteroid, however, suggest additional factors may be required to mediate endogenous, perhaps isoform-specific, differences in EcR conformation, consistent with previously reported effects of UpRXR isoforms on UpEcR ligand-binding affinities.