A New Perspective in Lernaea Cyprinacea Control International

International Journal of Biological Macromolecules 111 (2018) 614–622

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Chitosan-silver nanocomposites in goldﬁsh aquaria: A new perspective in Lernaea cyprinacea control☆

Nermeen M. Abu-Elala a,⁎, Marwa M. Attia b, Reham M. Abd-Elsalam c a Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Cairo University, Egypt b Department of Parasitology, Faculty of Veterinary Medicine, Cairo University, Egypt c Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Egypt article info abstract

Article history: Nanomedicine is a promising new research area in human and veterinary science. Metal nanoparticles Received 18 October 2017 have shown high biocidal activity against bacteria, fungi and viruses, few studies have focused on antiparasitic Received in revised form 3 December 2017 action. Therefore, this study aims to investigate the influence of chitosan-silver nanocomposites on the fish Accepted 26 December 2017 crustacean parasite Lernaea cyprinacea. The disease was detected in goldfish (Carassius auratus) aquaria during Available online 29 December 2017 the spring. Molecular and morphometric characterizations of the parasite were performed using polymerase chain reaction for rRNA and scanning electron microscopy. Chitosan-silver nanocomposites were characterized Keywords: Goldfish using transmission electron microscopy and Zetasizer. Probit analysis of parasite mortality versus the logarithmic Lernaea cyprinacea concentrations of the composites indicated that the 1 h/LC50 was 5.495 ppm. Parasites exposed to the chitosan- Chitosan-silver nanocomposite silver nanocomposites showed severe pathological alterations and adsorbed the composite particles on their Pathology cuticles. After aqueous exposure of the infected fish to the compound at its LC50 for 24 h, the female lernaeids PCR were completely dislodged. Moreover, the pathological findings indicated rapid skin wound healing and renewal and SEM at the parasitic injury site. Therefore, we concluded that chitosan-silver nanocomposites are potential parasitic control agents for ornamental glass aquaria, as they have detrimental effects on aquatic predators, such as copepods. © 2018 Elsevier B.V. All rights reserved.

1. Introduction of secondary bacterial and fungal infections that can eventually cause death [3,4]. Mechanically removing the parasitic female is impractical Lernaea cyprinacea (anchor worm) is the most abundant freshwater on a large scale, as the removal process is sometimes incomplete and crustacean copepod and is associated with many aquatic hosts (e.g., fish the anchor remains inside the fish tissue. Potassium permanganate and tadpoles) over a wide geographical range [1,2]. Members of the and organophosphate pesticides are the most commonly used Cyprinidae family, including koi, goldfish, and common carp, are the chemicals against L. cyprinacea; however, they typically affect the para- more commonly affected fish [3]. Only adult female lernaeids parasitize sitic stages that survive off the host more than they affect the adult the fish skin, gills, fins, eyes, mouth cavity and nostrils, but they appear stages, and precautions against possible fish toxicity should be consid- to prefer the abdomen and the base of the fins [4]. They have long fila- ered [5]. Several chemical therapy regimens against this copepod must mentous bodies with two trailing egg sacs, and their mouths and two be applied once per week for four weeks to ensure that all parasitic pairs of anchors embed into the fish musculature. Mass fish mortalities stages are completely eradicated [5]. Therefore, novel antimicrobial due to L. cyprinacea infestation are uncommon, but recurrent infections agents that control L. cyprinacea infection are required. The science of and long-term exposure decrease the growth performance of fish and nanomedicine applies nanoparticles in human and veterinary medicine, adversely affect their general appearance and the health status. Individ- and the use of nanoparticles in fish medicine is a promising new re- ual parasites can cause severe focal damage to the affected tissue includ- search area [6]. Metallic nanoparticles are used in numerous pharma- ing haemorrhagic and ulcerated lesions. These lesions increase the risk ceutical applications. Among all the antimicrobial metals, silver is well known for its strong toxicity against a wide range of microorganisms [7]. Silver nanoparticles (Ag NPs) have a high surface-to-volume ratio, which increases their biocidal action compared to that of their bulk ☆ The authors declare that they have no conflicts of interest. equivalent. Several mechanisms have been elucidated to explain the ⁎ Corresponding author at: Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Cairo University, Giza 11221, Egypt. biocidal effect of Ag NPs [7]. Chitosan is a biopolymer used to disperse E-mail address: [email protected] (N.M. Abu-Elala). and stabilize Ag NPs. The composites of Ag NPs with chitosan improve

https://doi.org/10.1016/j.ijbiomac.2017.12.133 0141-8130/© 2018 Elsevier B.V. All rights reserved. N.M. Abu-Elala et al. / International Journal of Biological Macromolecules 111 (2018) 614–622 615 the antimicrobial activities of Ag NPs at lower concentrations [8]. Cur- directions. Partial nucleotide sequences for both genes were submitted rently, most research focuses on the antibacterial and antifungal activi- to the National Center for Biotechnology Information (NCBI). ties of chitosan-silver nanocomposites, but few studies have focused on their antiparasitic action. 2.4. Chitosan-silver nanocomposites preparation Whether chitosan silver nanocomposites have biocidal effects against L. cyprinacea remains unknown. In this research, we proposed Chitosan from shrimp shells with an 85% degree of deacetylation a new method to control L. cyprinacea using this nanocomposites. L. was purchased from Sigma-Aldrich (Germany). Ag NPs with an average cyprinacea was identified by molecular methods and scanning electron particle size of 40–60 nm were purchased from NanoTech (Egypt). To microscopy (SEM), chitosan-silver nanocomposites were synthesized prepare the chitosan-silver nanocomposites, a 1 wt% chitosan solution and characterized by transmission electron microscopy and zetasizer. was prepared in 2% acetic acid. Ag NPs were dispersed in ethanol by Finally, the in vitro and in vivo antiparasitic effects of the chitosan-silver sonication for 20 min then added drop wise at a ratio of 5 wt% (with re- nanocomposites against L. cyprinacea were investigated. spect to chitosan) while stirring. The mixture was stirred for 4 h and sonicated for 30 min and then precipitated in a mixture of NaOH solu- 2. Materials and methods tion and ethanol. Next, the precipitate was filtered and washed several times with deionized water until a colourless filtrate was obtained 2.1. Fish examination and sampling with a neutral pH; this filtrate was then vacuum dried at 60 °C.

In March 2016, a local goldfish retailer in the Al-Sharqia governorate, 2.5. Characterization of the chitosan-silver nanocomposites Egypt suffered a heavy infestation of hairy worms (anchor worms). Dur- ing the investigation, the clinical history, signs, prevalence, and intensity The chitosan-silver nanocomposites were characterized by trans- of the infection were recorded. Approximately one hundred-twenty in- mission electron microscopy. Imaging was performed using a JEM- fected fingerlings were captured and transported to the laboratory in a 2100 (JEOL, Japan) operating at 80 kV, and the sample was sonicated 50 L tank with aeration. Adult and live parasitic copepods were mechan- in ethanol, deposited onto a copper-coated carbon grid, and allowed ically removed from the fish bodies and identified and characterized in to evaporate [14]. The particle size distribution and zeta potential laboratory, and the LC50 of the chitosan-silver nanocomposites was were measured using a Zetasizer (Nano-ZS, Malvern, UK). evaluated in vitro. 2.6. Determination of chitosan-silver nanocomposites LC50 on L. 2.2. Parasitological examinations cyprinacea

To study their morphology in detail, the copepods were washed sev- Two-fold serial dilutions (25 ppm, 12.5 ppm, 6.25 ppm, 3 ppm, eral times with saline then cleared in lactophenol and mounted in gela- 2 ppm, 1 ppm and 0.5 ppm) of the chitosan-silver nanocomposites tine [9]. The parasites were identified according to Markewitch [10]. were prepared using phosphate buffered saline as the solvent.L. Approximately five freshly collected copepods were first washed sev- cyprinacea-infested fish were euthanized with MS-222 to facilitate me- eral times with saline, and the fresh L. cyprinacea specimens were im- chanically removing the live female lernaeids. A total of 210 adult live L. mersed in 2.5% glutaraldehyde according to the method reported by cyprinacea parasitic females were collected and divided as 30 L. Robinson and Avenant-Oldewage [11]. Specimens were then cyprinacea/dilution/replicate. After a 1 h exposure to the graded chito- dehydrated through an ascending ethanol series, dried in a CO2 critical san-silver nanocomposites dilutions, the parasitic copepods were ex- point drier (Autosamdri-815, Germany), glued over stubs and coated amined under a light microscope, and mortality was recorded. with 20 nm of gold using a sputter coater (Spi-Module sputter coater, UK). Finally, the specimens were examined and photographed using 2.7. Demonstrating the effect of the chitosan-silver nanocomposites on L. SEM at magnifications ranging from 35× to 500× (JSM 5200, Electron cyprinacea using SEM Probe Microanalyzer, JEOL, Japan; at the Faculty of Agriculture, Cairo University). Five freshly dead L. cyprinacea parasitic females exposed to chitosan- silver nanocomposites were collected, washed several times with saline, 2.3. PCR identification and partial sequences of 18S and 28S rRNA genes and processed for SEM.

Five parasitic copepods were stored in absolute ethanol for molecu- 2.8. In vivo parasiticidal efficacy of the chitosan-silver nanocomposites lar analysis. Genomic DNA was extracted using a Qiagen DNA extraction kit following the manufacturer's instructions. The DNA quality and Fifteen goldfish fingerlings (weight 12.8 ± 2.2) infested with adult L. quantity were measured using a Thermo Scientific Nanodrop. Two cyprinacea were allocated in 10 L of dechlorinated tap water in glass sets of primers were used to amplify the 18S and 28S rRNA partial aquariums measuring 20 cm * 30 cm * 40 cm. They were maintained gene sequences (Table 1). PCR was performed as described by Song et under laboratory conditions for three days prior to exposure (22.0 ± 2 al. [12] and Stavrescu-Bedivan et al. [13] by initial denaturation at 94 °C, pH 6.9 ± 0.4, 6.0–7.8 mg L−1 dissolved oxygen). Fish were exposed °C for 5 min, followed by 30 cycles at 94 °C for 30 s, 54 °C for 30 s and to 5.495 ppm of the chitosan-silver nanocomposites in water for 24 h. 72 °C for 1 min with a final extension at 72 °C for 5 min. The PCR prod- Then, the water was siphoned out and replaced with fresh water. The ucts were visualized via agarose gel electrophoresis and Sanger se- fish were monitored for two weeks post-exposure, and they were fed quenced using a Sanger DNA sequencer (Applied Biosystems) in two once daily at 1% body weight with commercial fish pellet feed during this period. Control positive not treated and control negative groups were also monitored during the treatment experiment. All Institutional Table 1 and National Guidelines for the care and use of fisheries were followed. Primers used in this study.

Gene name Primers Product size 2.9. Histopathological examination of fish 18S-rRNA F-5′ AGGGTGTGMCCTATCAACT-3′ 1457 bp R-5′ TTACTTCCTCTAAACGCTC-3′ Tissue samples from the skin, fins, gills, and underlying muscles of 28S-rRNA F-5′ ACAACTGTGATGCCCTTAG-3′ 754 bp the infested fish were collected before and after aqueous exposure to R-5′ TGGTCCGTGTTTCAAGACG-3′ the chitosan-silver nanocomposites. Fish were euthanized using 616 N.M. Abu-Elala et al. / International Journal of Biological Macromolecules 111 (2018) 614–622

(Tricane methane sulfonate, MS-222 10 mg/L) before collecting tissue range from 2 to 5 mm in length (mean, 3.6 ± 0.4 mm). The worms samples. Tissues were fixed in 10% neutral buffered formalin, embedded have six pairs of swimming legs, with the first pair on the ventral side in paraffin and stained with haematoxylin and eosin (H&E) according to of the cephalothorax, the 2nd, 3rd and 4th pairs in the thorax, and the the methods described by Bancroft and Gamble [15]. 5th and 6th pairs in the abdomen.

2.10. Statistical analysis 3.3. Molecular identiﬁcation and nucleotide sequence accession numbers

Data were analysed using SPSS version 21. The 50% lethal concentra- Fig. 2E shows the PCR for the 18S rRNA and 28S rRNA partial gene se- tion with 95% confidence intervals was determined by probit analysis quences isolated from L. cyprinacea. Multiple sequence alignment re- [16,17]. The LC50 was calculated by the obtained regression equation sults indicated 99–100% identity with other recorded L. cyprinacea in (Y = mortality %; X = log concentration) or by drawing a transverse GenBank. Both genes were deposited in GenBank under the following line pass from probit 0.5 on the y-axis, then moving to the x-axis and accession numbers: KX258625 for 18S rRNA and KX258626 for 28S finding the associated log concentration, which was then inversed to rRNA. determine the LC50. 3.4. Characterization of the chitosan-silver nanocomposites 3. Results Fig. 3A shows the transmission electron microscopic image of the 3.1. Clinical examination chitosan-silver nanocomposites, revealing that the spherical Ag NPs ranging from 40 to 60 nm are embedded inside and around the chitosan Goldfish broodstocks and fingerlings have a history of recurrent infec- macromolecule changing its overall morphology. Fig. 4A shows the tions, particularly in the spring. Parasitic L. cyprinacea females appeared as particle size distribution curve obtained by DLS; the average particle grey-green hairy worms attached to focal areas along the sides of the host, size is 217 nm with a PdI of 0.335. The zeta potential of our compound on the flank region, behind the operculum, and on the dorsal and tail fins is +27 ± 4 mV (Fig.4B). (Fig. 1B, C). Focal reddish ulcers were also observed (Fig. 1A). The parasitism indices of L. cyprinacea are presented in Table 2. 3.5. Probite analysis of the chitosan-silver nanocomposites

3.2. Morphological criteria of adult copepods Fig. 3B shows the mortality against the logarithmic concentration of the chitosan-silver nanocomposites after a 1 h exposure period. The Adult female copepods are elongated and cylindrical, with a body LC50 was statistically determined by probit analysis to be 5.495 ppm. length of 9–15 mm (mean, 11 ± 0.7 mm) (Fig. 2A). The anchor has four horns. The ventral pair of horns is simple, while the dorsal pair is 3.6. SEM of L. cyprinacea after chitosan-silver nanocomposites exposure Y-shaped, composed of anterior and posterior parts (Fig. 2BandC). The mouth is small and is located between the two sets of horns. Fe- SEM examination of L. cyprinacea exposed to the chitosan-silver males have a cylindrical neck that gradually enlarges posteriorly. The nanocomposites shows severe irreversible alterations to the parasite's abdomen is short and round with two cylindrical, elongated egg sacs entire body, including marked anchor degeneration, wrinkles and pointed distally, containing several rows of eggs (Fig. 2D). Egg sacs marked longitudinal folds and distorted mouth portions (Fig. 5A).

Fig. 1. Goldfish (C. auratus) showing (A) focal haemorrhagic ulcers distributed in the skin behind the operculum, at the base of dorsal fin, and at the area of caudal peduncle. (B) L. cyprinacea parasitic females attached on the skin and the tail fin. (C) Two L. cyprinacea parasitic females attached together in one ulcer at the base of dorsal fin. (D) Healed ulcers at the base of dorsal fin. N.M. Abu-Elala et al. / International Journal of Biological Macromolecules 111 (2018) 614–622 617

Table 2 Parasitism indices of L. cyprinacea in goldﬁsh.

Fish No. of examined ﬁsh No. of parasitized ﬁsh Prevalence No. of parasites collected Intensity of parasitism

Carassius auratus 120 107 89.17% 245 2.29

Marked swellings appeared in the dermal layer of the trunk region 3.7. In vivo treatment findings (Fig. 5D) with a severely corrugated and shrunken carapace, leading to drastic changes in its morphology (Fig. 5A and B). Fissured areas along After two days, parasitic L. cyprinacea females had completely the trunk (Fig. 5C), and degenerated swimming legs were also observed. dislodged from the infected fish after exposure to the chitosan-silver The egg sac lost its smooth texture and configuration and appeared nanocomposites. The remaining ulcers appeared to be healed; they shrunken with nanocomposites aggregates surrounding it (Fig. 5EandF). were covered with a white membrane one week after exposure

Fig. 2. Examination of L. cyprinacea under SEM (A, B, C, D). (A) The elongated and cylindrical female copepod. (B& C) the ventral pairs of horns are simple while the dorsal pairs are Y- shape, composed from anterior and posterior part, the anchor has four horns. (D) Its abdomen is very short and rounded has two cylindrical elongated egg sacs pointed distally containing several rows of eggs. (E) Agrose gel electrophoresis of polymerase chain reaction for ampliﬁed products; Lane 1 is DNA ladder 100 bp; Lane 2 is L. cyprinacea 18s r RNA 1457 bp and Lane 3 is L. cyprinacea 28s r RNA 754 bp. 618 N.M. Abu-Elala et al. / International Journal of Biological Macromolecules 111 (2018) 614–622

Fig. 3. (A) TEM image of chitosan silver nanocomposites showing the spherical silver nanoparticles in the range of 40–60 nm are embedded inside and around the chitosan micro molecule. (B) Probite analysis to determine the 1h-LC50 of chitosan -silver nanocomposites against L. cyprinacea.

(Fig. 1D). The treated fish were apparently healthy, with no cases of re- dermis, the anterior anchor of the parasite was surrounded by fine infection during the monitoring period. connective tissue capsules (Fig. 6 A, C) and inflammatory cells. The dermal layer showed severe inflammation with extensively dilated 3.8. Histopathological results blood vessels, haemorrhaging (Fig. 6D), oedema and an intense leucocytic infiltration with neutrophils, macrophages and granular Histopathological examination of the skin and fins from the non- acidophilic cells (Fig. 6C, D). The underlying muscular layer revealed treated group infested with L. cyprinacea revealed severe damage to severe muscular necrosis, fragmentation with leucocytic infiltration the epidermal layer (Fig. 6B) at the parasitic penetration site. More- and bacterial aggregation. The gills showed mild necrosis of the re- over, the epithelium lining the other areas was hyperplastic. In the spiratory epithelium.

Fig. 4. (A) the particle distribution curve of chitosan silver nanocomposites (B) Zeta potential curve of chitosan silver nanocomposites. N.M. Abu-Elala et al. / International Journal of Biological Macromolecules 111 (2018) 614–622 619

Fig. 5. Examination of L. cyprinacea exposed to Chitosan silver nanocomposites under SEM shows (A) distortions and shrunken mouth portions (B) adsorption of chitosan silver nanocomposites on the cuticle as well as corrugated and shrunken carapace that affecting its morphological configuration. (C) Some fissured areas present on the trunk. (D) Marked swellings appeared in the dermal layer at the trunk region. (E&F) The egg sac loss its smooth texture with little configurations and shrunken with aggregation of nanocomposites around it.

The chitosan-silver nanocomposites-treated group showed mark- identities with Japanese, Chinese and Iranian isolates [12]. edly reduced inflammation in the skin, fins and underlying muscle Morphometrical SEM images revealed the Y-shaped dorsal arms of the layers. The epidermis showed marked proliferation of epithelial, muco- L. cyprinacea [11]. The antiparasitic activity of the chitosan-silver nano- sal and alarm cells (Fig. 7A). The wound surface showed approximated composites was evaluated on L. cyprinacea. The particle size and zeta wound edges with surface re-epithelization. The dermal layer showed potential of the nanocomposites influence their antimicrobial activities extensive fibroplasia with well-organized collagen fibre formation [20,21]. Marked pathological alterations were observed in L. cyprinacea (Fig. 7B, C). Angiogenesis was also observed with newly formed blood exposed to the chitosan-silver nanocomposites at LC50 for 1 h. Nano- capillaries (Fig. 7C). The underlying muscular layer revealed mild de- particles were adsorbed over the entire body of the lernaeids, swelling generation with mild mononuclear cell aggregation. In some cases, the and fissures in the trunk and shrinkage in the eggs were observed. parasite's anterior anchor was atrophied and surrounded by a few Some studies have revealed nanoparticle adsorption on the exoskeleton chronic inflammatory cells (Fig. 7C). The gills showed mild to moderate of aquatic invertebrates [22,23]; however, the mechanism of interaction proliferation of the gill lamellae respiratory epithelium. between the nanoparticles and the cuticle is unclear. The crustacean cuticle is mostly composed of fibrous phase of crystalline chitin 4. Discussion (nanofibrils with diameters of 3 nm), sugars, silk-like proteins attached through specific H-bonds and globular proteins, which confer a net neg- L. cyprinacea causes serious problems in ornamental fish marketing ative surface charge at neutral pH [24,25] . A zeta potential of +27 mV [18]. The present study demonstrated a heavy L. cyprinacea infestation was measured on the surface of chitosan-silver nanocomposites, corre- in goldfish glass aquaria with a high prevalence (89.17%) and intensity sponding to a global positive charge, which generates a potential attrac- ratio (2.29). Female lernaeids appeared attached to the skin of the tion between the nanocomposites and the cuticle. The polycationic flank region and base of the fins, as these sites offer greater protection chitosan has an affinity for the surface of the negatively charged cuticle against water currents [4,19]. Molecular identification and multiple se- and affects the cells' osmotic stability, resulting in subsequent leaking of quence alignments of 18S and 28S rRNA genes revealed 99–100% cellular constituents. Meanwhile, small Ag NPs can anchor onto the 620 N.M. Abu-Elala et al. / International Journal of Biological Macromolecules 111 (2018) 614–622

Fig. 6. The histopathological finding in non-treated Lernea cyprinaceae infested group. (A) The Lernea cyprinaceae parasite surrounded by connective tissue capsule (arrow) (H&E ×200); (B) Skin showing destruction of the epidermal layer (arrow) with vacuolization of the remaining epidermal cells and dermal inflammatory cell infiltration (H&E ×200); (C) The dermis showing Lernea cyprinaceae (arrow) surrounding by fine connective tissue capsule associated with dense aggregation of neutrophils, macrophage and granular acidophilic cells (H&E ×400); (D) Dermis showing severe dilatation of the dermal blood vessels with intense inflammatory cell aggregation (H&E ×200). microbial cell wall and penetrate it. The pits formed by Ag NPs on the formed by Ag NPs can damage the cell membrane, making it porous cell surface increase the accumulation of Ag NPs on the surface. The sil- [26,27]. ver ions (Ag+) released by the Ag NPs can then interact with the thiol Histopathological examination of the non-treated L. cyprinacea- groups of vital respiratory enzymes and inactivate them. Free radical infested fish revealed destruction of the epidermal layer, severe dermal

Fig. 7. The histopathological finding in the chitosan-silver nanocomposites treated L. cyprinaceae infested group. (A) Epidermis showing marked hyperplasia of the epithelial, mucous (arrow) and alarm cells (arrow head) (H&E ×200); (B) Skin showing epidermal hyperplasia with fibroblast and angioblast proliferation, fibrin and inflammatory cell aggregation in the dermal tissue (H&E ×200); (C) The dermis showing organized fibroblast, newly formed blood capillaries, angioblast proliferation and mononuclear cell infiltration (H&E ×200); (D) Muscle showing atrophied Lernea cyprinaceae anterior anchor (arrow) with mild inflammatory cell aggregation (H&E ×200). N.M. Abu-Elala et al. / International Journal of Biological Macromolecules 111 (2018) 614–622 621 inflammation and muscular necrosis, consistent with the results previ- [5] A.E. Goodwin, Massive Lernaea cyprinacea infestations damaging the gills of channel catfish polycultured with bighead carp, J. Aquat. Anim. 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