Shining a light on parasite behaviour: daily patterns of Argulus fish lice.

Parasites display a wide range of behaviours that are frequently overlooked in favour of host responses. Understanding these behaviours can improve parasite control through a more precise application or development of new behaviour-based strategies. In aquaculture fish lice are an ongoing problem, infections reduce fishery production and control options are limited. Fish lice are distinct in their ability to survive and swim off hosts, allowing the transmission to multiple fish hosts across their lifespan. Here we assessed the off-host behaviour of Argulus foliaceus (a freshwater fish louse) and observed a diurnal rhythmical pattern in their behaviour. This pattern was lost when lice were exposed to constant darkness, indicating that the behaviour is not endogenously driven. Males were consistently active in light with reduced activity in darkness. In contrast, females were active during light and dark phases with peak activity at the start of dark periods. A. foliaceus was also strongly attracted to a light stimulus, preferring white- and blue-coloured lights over green- or red-coloured lights. Light is a strong driver of fish louse activity and could be used to trap parasites. Aquaculture light regimes could also be altered to reduce parasite attraction and activity.

Bet hedging and cold-temperature termination of diapause in the life history of the Atlantic salmon ectoparasite Argulus canadensis.

Argulus canadensis is a crustacean ectoparasite observed increasingly on wild migrating adult Atlantic salmon. We investigated temperature and salinity tolerance regarding development, survival and hatch of A. canadensis eggs to help understand spatiotemporal features of transmission. Argulus canadensis eggs differentiate to pharate embryos by 35 days buttheir hatch is protracted to ~7 months. Cold treatment ⩾75 days mimics overwintering and terminates egg diapause, with 84.6% (72.1-100%) metanauplius hatch induced ⩾13 °C and synchronized to 3-4 weeks. Inter- and intra-clutch variability and protracted hatch in the absence of cold-temperature termination of diapause is compatible with bet hedging. Whereas diapause likely promotes phenological synchrony for host colocalization, bet hedging could afford temporal plasticity to promote host encounter during environmental change. Our egg storage and hatch induction/synchronization methodologies can be exploited for empirical investigations. Salinity tolerance reveals both significantly higher embryonic development (94.4 ± 3.5% vs 61.7 ± 24.6%) and metanauplius hatch (53.3 ± 7.5% vs 10.1 ± 8.2%) for eggs in freshwater than at 17 ppt. Unhatched embryos were alive in freshwater by the end of the trial (213 days) but were dead/dying at 17 ppt. Eggs did not develop at 34 ppt. Salinity tolerance of A. canadensis eggs supports riverine transmission to adult Atlantic salmon during return to freshwater for mating each year.

Stable isotopes reveal contrasting trophic dynamics between host-parasite relationships: A case study of Atlantic salmon (Salmo salar) and parasitic lice (Lepeophtheirus salmonis and Argulus foliaceus).

Across existing fish host-parasite literature, endoparasites were depleted in δ15 N compared to their hosts, while ectoparasitic values demonstrated enrichment, depletion and equivalence relative to their hosts. δ13 C enrichment varied extensively for both endo- and ectoparasites across taxa and host tissues. In our case study, sea lice (Lepeophtheirus salmonis) were enriched in δ15 N relative to their farmed Atlantic salmon (Salmo salar) hosts, although the value contradicted the average that is currently assumed across the animal kingdom. Common fish lice (Argulus foliaceus) did not show a consistent trend in δ15 N compared to their wild S. salar hosts. Both parasitic species had a range of δ13 C enrichment patterns relative to their hosts. Farmed and wild S. salar had contrasting δ13 C and δ15 N, and signals varied across muscle, fin and skin within both groups. L. salmonis and A. foliaceus subsequently had unique δ13 C and δ15 N, and L. salmonis from opposite US coasts differed in δ15 N. Given the range of enrichment patterns that were exhibited across the literature and in our study system, trophic dynamics from host to parasite do not conform to traditional prey to predator standards. Furthermore, there does not appear to be a universal enrichment pathway for δ13 C nor δ15 N in parasitic relationships, which emphasizes the need to investigate host-parasite linkages across species.

Life in the fast lane: Temperature, density and host species impact survival and growth of the fish ectoparasite Argulus foliaceus.

With expanding human populations, the food sector has faced constant pressure to sustainably expand and meet global production demands. In aquaculture this frequently manifests in an animal welfare crisis, with fish increasingly farmed under high production, high stress conditions. These intense environments can result in fish stocks having a high susceptibility to infection, with parasites and associated disease one of the main factors limiting industry growth. Prediction of infection dynamics is key to preventative treatment and mitigation. Considering the climatic and technology driven changes facing aquaculture, an understanding of how parasites react across a spectrum of conditions is required. Here we assessed the impact of temperature, infection density and host species on the life history traits of Argulus foliaceus, a common palearctic fish louse, representative of a parasite group problematic in freshwater aquaculture and fisheries worldwide. Temperature significantly affected development, growth and survival; parasites hatched and developed faster at higher temperatures, but also experienced shorter lifespans when maintained off the host. At high temperatures, these parasites will likely experience a short generation time as their life history traits are completed more rapidly. A. foliaceus additionally grew faster on natural hosts and at lower infection densities. Ultimately such results contribute to prediction of population dynamics, aiding development of effective control to improve animal welfare and reduce industry loss.

Crustacean parasites of Leporinus affinis, an Anostomidae fish from the Brazilian Amazon.

This study investigated the fauna of ectoparasitic crustaceans in Leporinus affinis from Reservoir Coaracy Nunes, in eastern Amazon (Brazil), as well as the parasite-host interactions. The mouth, gills and tegument of 50.9% of the fish examined were parasitized by Argulus chicomendesi, Ergasilus turucuyus and Excorallana berbicensis, and a total of 118 parasites were collected. The dominance was found for E. berbicensis and the higher infestation levels were caused by E. berbicensis on the body surface of the hosts, but E. turucuyus had the highest prevalence on the gills of this host. The cluster analysis revealed higher similarity in the infestations by E. berbicensis and A. chicomendesi in relation to infestation site in hosts. Host sex and relative condition factor (Kn) were not influenced by moderate parasitism, but the abundance of parasites presented negative correlation with weight and Kn of the fish. This is the first study on the parasites of L. affinis showing low species diversity, with moderate prevalence and low parasite abundance.

Crustacean parasites of Leporinus affinis, an Anostomidae fish from the Brazilian Amazon / Crustáceos parasitos de Leporinus affinis, um Anostomidae da Amazônia brasileira

Abstract This study investigated the fauna of ectoparasitic crustaceans in Leporinus affinis from Reservoir Coaracy Nunes, in eastern Amazon (Brazil), as well as the parasite-host interactions. The mouth, gills and tegument of 50.9% of the fish examined were parasitized by Argulus chicomendesi, Ergasilus turucuyus and Excorallana berbicensis, and a total of 118 parasites were collected. The dominance was found for E. berbicensis and the higher infestation levels were caused by E. berbicensis on the body surface of the hosts, but E. turucuyus had the highest prevalence on the gills of this host. The cluster analysis revealed higher similarity in the infestations by E. berbicensis and A. chicomendesi in relation to infestation site in hosts. Host sex and relative condition factor (Kn) were not influenced by moderate parasitism, but the abundance of parasites presented negative correlation with weight and Kn of the fish. This is the first study on the parasites of L. affinis showing low species diversity, with moderate prevalence and low parasite abundance.

Resumo Este estudo investigou a fauna de crustáceos parasitos de Leporinus affinis do Reservatório Coaracy Nunes, na Amazônia oriental (Brasil), bem como a interação parasito-hospedeiro. A boca, brânquias e tegumento de 50.9% dos peixes examinados estavam parasitados por Argulus chicomendesi, Ergasilus turucuyus e Excorallana berbicensis, e um total de 118 parasitos foram coletados. A dominância foi de E. berbicensis, e os maiores índices de infestação foram causados por E. berbicensis na superfície corporal dos hospedeiros, mas E. turucuyus teve a maior prevalência nas brânquias. Análises de grupamento evidenciaram maior similaridade no padrão de infestação de E. berbicensis e A. chicomendesi quanto ao sítio de infestação nos hospedeiros. O sexo dos hospedeiros e fator de condição relativo (Kn) não foram influenciados pelo moderado parasitismo, mas a abundância de parasitos apresentou correlação negativa com o peso e Kn dos peixes. Este é o primeiro estudo sobre parasitos de L. affinis, mostrando baixa diversidade de espécies, com moderada prevalência e baixa abundância parasitária.

Identification and functional characterization of a g-type lysozyme gene of Labeo rohita, an Indian major carp species.

Lysozyme, an important secretory innate immune component, possesses antimicrobial activity against broad spectrum of bacteria and viruses. In the present study, complete CDs (558 bps) of g-type lysozyme of rohu (Labeo rohita) was amplified and translated for a putative protein of 185 amino acids. The domain architecture and tertiary structure was also predicted for the protein. Its expression profile was studied in three infection models (bacteria: Aeromonas hydrophila, poly I:C, a dsRNA viral analogue and an ectoparasite: Argulus siamensis) in liver and kidney tissues of rohu. An up-regulation of 630-fold and 420-fold of the gene was observed at 48 h in liver and anterior kidney tissues respectively, after A. hydrophila infection. Significant increase in transcript level was noticed in both liver (0.8-fold) and kidney (480-fold) after 1 h and 12 h of poly I:C induction, respectively. Similarly, expression of lysozyme g transcripts was increased 6000-fold after 7 d of A. siamensis infection in liver tissue. The recombinant protein of g-type lysozyme of rohu (rLr-lysG) of 20.19 kDa was produced in Escherichia coli system and the lysozyme activity of rLr-lysG was found to be most active at pH 6.0 and temperature 35 °C. The potential lytic activity was found to be against A. hydrophila (UL = 0.53) followed by for E. tarda (UL = 0.45) whereas the lytic activity was the least against S. aureus (UL = 0.35) and M. lysodeikticus (UL = 0.34), at pH 6.0 and temperature 35 °C. The normal serum level of protein was estimated using indirect ELISA and was found to be very low (0.12-0.15 µg/ml). These results suggested that g-type lysozyme of rohu might be a potent immunostimulant against microbial infections, with a major role in innate immunity.

Parasitic sea louse infestations on wild sea trout: separating the roles of fish farms and temperature.

BACKGROUND: The causal relation between parasitic sea lice on fish farms and sea lice on wild fish is a controversial subject. A specific scientific debate has been whether the statistical association between infestation pressure (IP) from fish farms and the number of parasites observed on wild sea trout emerges purely because of a confounding and direct effect of temperature (T). METHODS: We studied the associations between louse infestation on wild sea trout, fish farm activity and temperature in an area that practices coordinated fallowing in Nordhordland, Norway. The data were sampled between 2009 and 2016. We used negative binomial models and mediation analysis to determine to what degree the effect of T is mediated through the IP from fish farms. RESULTS: The number of attached lice on sea trout increased with the T when the IP from fish farms was high but not when the IP was low. In addition, nearly all of the effect of rising T was indirect and mediated through the IP. Attached lice remained low when neighbouring farms were in the first year of the production cycle but rose substantially during the second year. In contrast to attached lice, mobile lice were generally seen in higher numbers at lower water temperatures. Temperature had an indirect positive effect on mobile louse counts by increasing the IP which, in turn, raised the sea trout louse counts. Mobile louse counts rose steadily during the year when neighbouring farms were in the first year of the production cycle and stayed high throughout the second year. CONCLUSIONS: The estimates of the IP effect on louse counts along with the clear biennial pattern emerging due to the production cycle of fish farms clearly indicate that fish farms play an important role in the epidemiology of sea lice on wild sea trout. Furthermore, the mediation analysis demonstrates that a large proportion of the effect of T on louse counts is mediated through IP.

The cost of infection: Argulus foliaceus and its impact on the swimming performance of the three-spined stickleback (Gasterosteus aculeatus).

For fish, there can be multiple consequences of parasitic infections, including the physical impacts on swimming and the pathological costs of infection. This study used the three-spined stickleback (Gasterosteus aculeatus) and the ectoparasitic fish louse, Argulus foliaceus, to assess both physical (including form drag and mass) and pathological effects of infection. Both sustained (prolonged swimming within an open channel flume) and burst (C-start) swimming performance were measured on individual fish before (trials 1-2) and after infection (trials 3-5). Experimental infection occurred shortly before the third trial, when the physical impacts of infection could be separated from any subsequent pathology as transmission of adult parasites causes instantaneous drag effects prior to observable pathology. Despite the relatively large size of the parasite and corresponding increase in hydrodynamic drag for the host, there were no observable physical effects of infection on either sustained or burst host swimming. By contrast, parasite-induced pathology is the most probable explanation for reduced swimming performance across both tests. All sticklebacks displayed a preference for flow refugia, swimming in low-velocity regions of the flume, and this preference increased with both flow rate and infection time. This study suggests that even with large, physically demanding parasites their induced pathology is of greater concern than direct physical impact.

Chonopeltis australis (Crustacea: Branchiura); the female reproductive system.

The female reproductive system has been described for Dolops ranarum (Stuhlman, 1891) and various Argulus spp. but, there is no description of the reproductive system for Dipteropeltis spp. Calman, 1912 or Chonopeltis spp. Thiele, 1900. This paper describes the female reproductive system and egg laying behaviour of Chonopeltisaustralis Boxshall, 1976 using histology, light microscopy, scanning electron microscopy and observations. The histological study of six specimens showed that the organ structure is similar to that of Argulus spp. and D. ranarum. The oocytes therefore develop in the gonocoel, are ovulated into the lumen of the ovary which is continuous with the functional oviduct and eventually the gonopore. Females of C. australis deposit eggs on the surrounding substrate while the fish is at rest, without leaving the host, thereby mitigating the risk of not locating a host again.

Apolipoprotein A-I in Labeo rohita: Cloning and functional characterisation reveal its broad spectrum antimicrobial property, and indicate significant role during ectoparasitic infection.

Apolipoprotein A-I (ApoA-I) is the most abundant and multifunctional high-density lipoprotein (HDL) having a major role in lipid transport and potent antimicrobial activity against a wide range of microbes. In this study, a complete CDS of 771 bp of Labeo rohita (rohu) ApoA-I (LrApoA-I) encoding a protein of 256 amino acids was amplified, cloned and sequenced. Tissue specific transcription analysis of LrApoA-I revealed its expression in a wide range of tissues, with a very high level of expression in liver and spleen. Ontogenic study of LrApoA-I expression showed presence of transcripts in milt and 3 h post-fertilization onwards in the larvae. The expression kinetics of LrApoA-I was studied upon infection with three different types of pathogens to elucidate its functional significance. Its expression was found to be up-regulated in the anterior kidney of L. rohita post-infection with Aeromonas hydrophila. Similarly following poly I:C (poly inosinic:cytidylic) stimulation, the transcript levels increased in both the anterior kidney and liver tissues. Significant up-regulation of LrApoA-I expression was observed in skin, mucous, liver and anterior kidney of the fish challenged with the ectoparasite Argulus siamensis. Immunomodulatory effect of recombinant LrApoA-I (rApoA-I) produced in Escherichia coli was demonstrated against A. hydrophila challenge in vivo. L. rohita administered with rApoA-I at a dose of 100 µg exhibited significantly higher protection (∼55%) upon challenge with A. hydrophila 12 h post-administration of the protein, in comparison to that observed in control group, along with higher level of expression of immune-related genes. The heightened expression of ApoA-I observed post-infection reflected its involvement in immune responses against a wide range of infections including bacterial, viral as well as parasitic pathogens. Our results also suggest the possibility of using rApoA-I as an immunostimulant, particularly rendering protection against A. hydrophila.

Predicting the effectiveness of depth-based technologies to prevent salmon lice infection using a dispersal model.

Salmon lice is one of the major parasitic problems affecting wild and farmed salmonid species. The planktonic larval stages of these marine parasites can survive for extended periods without a host and are transported long distances by water masses. Salmon lice larvae have limited swimming capacity, but can influence their horizontal transport by vertical positioning. Here, we adapted a coupled biological-physical model to calculate the distribution of farm-produced salmon lice (Lepeophtheirus salmonis) during winter in the southwest coast of Norway. We tested 4 model simulations to see which best represented empirical data from two sources: (1) observed lice infection levels reported by farms; and (2) experimental data from a vertical exposure experiment where fish were forced to swim at different depths with a lice-barrier technology. Model simulations tested were different development time to the infective stage (35 or 50°-days), with or without the presence of temperature-controlled vertical behaviour of lice early planktonic stages (naupliar stages). The best model fit occurred with a 35°-day development time to the infective stage, and temperature-controlled vertical behaviour. We applied this model to predict the effectiveness of depth-based preventive lice-barrier technologies. Both simulated and experimental data revealed that hindering fish from swimming close to the surface efficiently reduced lice infection. Moreover, while our model simulation predicted that this preventive technology is widely applicable, its effectiveness will depend on environmental conditions. Low salinity surface waters reduce the effectiveness of this technology because salmon lice avoid these conditions, and can encounter the fish as they sink deeper in the water column. Correctly parameterized and validated salmon lice dispersal models can predict the impact of preventive approaches to control this parasite and become an essential tool in lice management strategies.

Variation in susceptibility pattern of fish to Argulus siamensis: Do immune responses of host play a role?

Branchiuran ectoparasites of the genus Argulus can have extensive damaging effects on cultured fish. There exist no systematic studies that evaluate susceptibility or resistance of various carp species to Argulus sp. and the underlying mechanisms. The present study aimed at identifying the most susceptible and resistant cultured species, studying settlement and survival of parasite on these species, and finally unravelling the variations of immune response in both resistant and susceptible species. Fish from eight species (Labeo rohita, Cirrhinus mrigala, Catla catla, Hypophthalmichthys molitrix, Cyprinus carpio, Ctenopharyngodon idella, Carassius auratus, Labeo fimbriatus) were individually challenged with metanauplii of A. siamensis (100 metanauplii/fish) before rearing them in single tank in triplicate for 45 days. Based on the observed parasite load on each species, L. rohita was found to be the most susceptible and C. idella the resistant species. The settlement and survival of the parasite on L. rohita and C. idella was compared at 24, 48, 72 and 96h post experimental infection. Survival was significantly low at 72h onwards in C. idella indicating it is an unsuitable/poorly preferred host for A. siamensis. The inflammatory responses which are known to be related to susceptibility were analysed. Individuals of both the species were exposed to A. siamensis (100 parasites/fish), and after 24h and 3 d, skin samples directly from the attachment site and non-attachment sites were assessed for transcriptomic profiles of selected innate defence genes. Artificial skin abrasion permitted comparisons between abrasion associated injury and louse-associated injury. The inflammatory responses varied significantly between both species indicating their role in determining susceptibility of a host to A. siamensis. The expression of major histocompatibility class II and matrix metalloproteinase 2 was significantly higher in C. idella compared to L. rohita and therefore appeared to be involved in the early protective response against A. siamensis. It is essential to study the expression pattern of more participatory genes of the inflammation related pathways to understand species specific susceptible patterns.

Mutualistic association of rotifer Philodina roseola with the branchiuran fish ectoparasite Argulus bengalensis at its embryonic stage.

Several rotifers including Philodina spp. are well known to make commensal and parasitic associations with different animals. The present investigation was carried out to decipher the relationship of Philodina roseola with a piscine ectoparasite Argulus bengalensis in its embryonic stage. Mechanical removal of the symbiont P. roseola from the argulid egg strips resulted in the complete (100%) failure in hatching. Several P. roseola individuals were found to feed on the solidified jelly coat of the eggs enabling the larvae to emerge under both laboratory and field conditions. Under the laboratory condition, the experimental removal of P. roseola did not affect the embryonic development, but it rendered the jelly coat intact; therefore, the larvae were unable to make hatching furrow and subsequently died. The results of our experiments thus prove this service-resource relationship to be a mutualism. Although the association is facultative for P. roseola, it is obligatory for Argulus spp. An act of intervention in this relationship thus offers a promising control of argulosis.

Transcriptional changes in three immunoglobulin isotypes of rohu, Labeo rohita in response to Argulus siamensis infection.

Immunoglobulin heavy chains of three isotypes viz., IgM, IgD and IgT/IgZ are described in teleosts. In this study, a challenge experiment with an ectoparasite Argulus siamensis was conducted to evaluate the changes in adaptive immune response by quantitation of expression of Ig heavy chains in skin, head kidney and mucus of infected rohu, Labeo rohita. Rohu were challenged with 100 metanauplii of A. siamensis/fish. Head kidney, skin and mucus samples were collected at 0 h, 12 h, 24 h, 3 d, 7 d, 15 d and 30 d by sacrificing four fish each from infected and control groups at each time point. The expression of IgM, IgD and IgZ in these tissues were measured by reverse transcription real time quantitative PCR. IgM level was found to reach its peak significantly 30 d post-infection in head kidney tissue, while IgM transcripts were below detectable range in skin and mucus at all time points. IgZ and IgD levels were significantly up-regulated post-infection in all the three tissue samples. Early up-regulation of IgD was observed in skin and mucus, compared to head kidney. This study showed that parasitic invasion can trigger varied expressions of immunoglobulin types to provide systemic as well as local protection in the host. In particular, the appearance of high level of expression of IgZ and IgD in skin and mucus will pave the way for vaccine development against A. siamensis which feeds on those tissues.

Behavioural adaptations of argulid parasites (Crustacea: Branchiura) to major challenges in their life cycle.

Fish lice (Argulus spp.) are obligate ectoparasites, which contrary to most aquatic parasites, retain the ability to swim freely throughout the whole of their life. In fish farms, they can quickly increase in numbers and without effective control cause argulosis, which results in the reduced growth and survival of their fish hosts. The morphology of Argulus spp, including their sensory organs, is suitable for both parasitism and free-swimming. By spending a considerable amount of time away from their host, these parasites risk being excessively dispersed, which could endanger mating success. Here we present a review of recent studies on the behaviour of Argulus spp, especially the aggregative behaviour that mitigates the dilution of the parasite population. Aggregation of parasites, which is especially important during the period of reproduction, occurs on different scales and involves both the aggregation of the host and the aggregation of the parasites on the host. The main behavioural adaptations of Argulus spp, including searches for hosts and mates, host manipulation and host choice, are all focused on the fish. As these ectoparasites repeatedly change hosts and inflict skin damage, they can act as vectors for fish pathogens. The development of environmentally friendly measures for the control and prevention of argulosis needs to take into account the behaviour of the parasites.

Chonopeltis australis (Crustacea) male reproductive system morphology; sperm transfer and review of reproduction in Branchiura.

The morphology of the male reproductive system as well as sperm transfer in Branchiura has been described for Dolops ranarum and Argulus japonicus. In this study, the reproductive system and accessory structures are described for male Chonopeltis australis using histology, light microscopy, and scanning electron microscopy. For the first time, we describe sperm transfer by means of a spermatophore in this genus. The internal and external morphology and mechanism of sperm transfer is compared with other Branchiura, where it has been described. The morphology of the reproductive system of C. australis is similar to that of D. ranarum while the accessory structures and the spermatophore produced are similar to that of A. japonicus. A revision of the definition of Branchiura with respect to reproduction is provided.

Molecular characterization of interleukin 15 mRNA from rohu, Labeo rohita (Hamilton): Its prominent role during parasitic infection as indicated from infection studies.

Interleukin 15 (IL-15) is an important cytokine of fish immune system. Sequence characterization of IL-15 from rohu, Labeo rohita revealed a mRNA sequence of 1064 bp with coding sequence of 567 bp and signal peptide of 16 amino acids. There are four characteristic sequence features viz., presence of four out-of-frame AUG initiation codons, four highly conserved cysteine residues, constitutive expression in all tissues and evolutionary similarity. The ontogeny study revealed maternal transfer of this molecule and higher expression up to 3 h post-fertilization in fertilized embryos. Its expression was down-regulated in anterior and posterior kidneys, intestine and liver tissues of rohu infected with Aeromonas hydrophila. Mild up-regulation in liver and higher expression in spleen was noticed in rohu stimulated with poly I:C (poly ionosinic:cytidylic), whereas down-regulation was observed in intestine and kidney tissues. However, a consistent higher expression was noticed in kidney and skin tissues during Argulus siamensis infection. Therefore, rohu IL-15 might possess more defensive role during early development and parasitic infection.

Effect of aqueous extract of Azadirachta indica A. Juss (neem) leaf on oocyte maturation, oviposition, reproductive potentials and embryonic development of a freshwater fish ectoparasite Argulus bengalensis Ramakrishna, 1951 (Crustacea: Branchiura).

In present study, a microcosm experiment is carried out to investigate the efficacy of 120 and 250-ppm crude aqueous extract of Azadirachta leaf on oocyte maturation, oviposition, embryonic development and hatching of the eggs of a fish ectoparasite Argulus bengalensis. Relative abundance of different maturing oocyte stages in the ovary of the parasite from different age groups was enumerated, and marked variations were obtained. Significant depletion in the abundance of pre-vitollogenic, vitellogenic and post-vitellogenic oocytes was recorded, which indicates impairment in maturation. Chromatin condensation of the oocytes of treated parasite indicates apoptosis of oogenic cells. Strong oviposition deterrence was evident by the elevated oviposition deterrence index of 0.18 and 0.52 at respective toxin levels. The treated parasites invested less number of eggs per oviposition, and hatching percentage of the eggs reduced markedly. In vitro treatment of eggs within 70 min of incubation exhibited coagulation of yolk material and subsequent reduction in hatching percentage. However, treatment applied after this critical period, hatching was not significantly altered. In vitro treatment of eggs at 80 min of incubation resulted in normal development. It signifies that azadirachtin affects the early developmental events but not the later. Presumably, azadirachtin either affects early gene expression of the embryo or antagonizes any of the substances of the zygote required for sustaining early developmental process. The result of the present experiment suggests that azadirachtin could be a promising agent to control argulosis through inhibition of the reproductive maturity of the parasite as well as through interference of its embryonic development.

First evidence of comparative responses of Toll-like receptor 22 (TLR22) to relatively resistant and susceptible Indian farmed carps to Argulus siamensis infection.

Toll-like receptor 22 (TLR22) is present in teleost but not in mammals. Among Indian farmed carps, Catla catla is relatively more resistant than Labeo rohita to Argulus siamensis lice infection. TLR22 is believed to be associated with innate immunity against ectoparasite infection. To investigate the TLR22 mediated immunity against argulosis, we have cloned and characterized TLR22 genes of L. rohita (rTLR22) and C. catla (cTLR22). The full-length cDNAs of rTLR22 and cTLR22 contained an open reading frame of 2838 and 2841 nucleotides, respectively; bearing the typical structural features. Phylogenetically rTLR22/cTLR22 was most closely related to Cyprinus carpio (common carp) counterpart, having highest sequence identity of 86.0%. The TIR domain remained highly conserved with 90% identity within freshwater fishes. The sequence information of cDNA and genomic DNA together revealed that the rTLR22/cTLR22 genes are encoded by uninterrupted exons. The co-habitation challenge study with A. siamensis infection confirmed that C. catla is comparatively more resistant than L. rohita. Further, comparative mRNA expression profile in immune relevant tissues also suggested about the participatory role of TLR22 during lice infection. However, TLR22 might not solely be involved in conferring relative resistance among carp species against argulosis.