Journal of the Egyptian Society of Parasitology, Vol.44, No.3, December 2014

J. Egypt. Soc. Parasitol. (JESP), 44(3), 2014: 627 – 637

BIOLOGICAL AND HEMATOLOGICAL RESPONSES OF ALEXANDRINA TO MYCOBIOSYNTHSIS SILVER NANOPARTICLES By HODA ABDEL-HAMID1 AND AMAL A.I. MEKAWEY2 Department OF Environmental Researches and Medical Malacology1, Theodor Bilharz Research Institute and Regional Center for Mycology and Biotechnology2, Al-Azhar University, Cairo, Egypt. Abstract Silver nanoparticles (AgPNs) extracts were prepared from seven Seven fungal isolates were evaluated through measuring their toxicity against Biomphalaria alexandrina . The effects of the two promising Paecilomyces variotii and Aspergillus niger AgNPs sub- lethal concentrations (LC10 & LC25) on the levels of steroid sex hormones, liver enzymes, total protein, lipids, albumin, glucose, total and differential count of hemocytes and mor- phology of hemocytes, oocytes and sperms were studied in this work. The short period of snails’ exposure (24h) to the two fungal AgNPs resulted in signifi- cant decrease in the levels of progesterone in B. alexandrina. The level of testosterone hormone showed significant increase in snails exposed to P. variotii AgNPs while no significant change was recorded at the exposure to A. niger AgNPs. Also, estradiol hor- mone concentration increased significantly in this investigation with the increase of the concentration of the two tested compounds. In addition, significant elevation in ALT, AST and Alkaline phosphatase was recorded. The total number of the hematocytes in- creased significantly by17.4-47.8%. Snails’ granulocytes were reduced by 19.1- 43.8%, while hyalinocytes increased by 63.6-354.5%. The exposure of B. alexandrina to LC25 of both P. variotii and A. niger AgNPs showed apoptotic hemolymph cells, fragmented, vacuolated and degenerated cytoplasm, shrunken nucleus and phagocytosis in the light microscopy photographs of the hemocytes. Besides, the photographs showed also, ab- normal nuclear division, degeneration and large fat vacuoles in the cytoplasm and swal- lowed atretic oocytes. Also, the photographs showed dead sperm head separated from its tail, other sperms showed abnormal swallowed head with severely nodded tail, dead sperms with wrinkled tails, hyperplasia and necrotic sperm heads led to overlapping of tails. In conclusion, applying the biosynthesized compounds which led to destruction of blood cells (the immune system), ova and sperms (the reproductive system) of snails is an important effective step to control schistosomiasis. Key words: Biomphalaria alexandrina, Paecilomyces variotii, Aspergillus niger, AgNPs, steroid hormones, granulocytes, hyalinocytes, schistosomiasis.

Introduction ducing or eliminating transmission and There are multiple parameters affect- remains among the methods of choice ing the transmission of schistosomiasis for schistosomiasis control (Rizk et al., as water related vector-borne disease. 2012). A new approach to control the Among them, the life expectancy of the vector snails of schistosomiasis; Bi- intermediate hosts, in which schis- omphalaria alexandrina is the using of tosome larvae undergo asexual repro- nanoparticles (El-Hommossany and El- duction and developed to the human Sherbibni, 2011). infecting stage “cercariae” (Anderson On the other hand, chemical and physi- and May, 1985). Snail control could be cal methods have various limitations in regarded as a rapid and efficient of re- terms of synthesis procedures which

627 may involve use of drastic experimental culating hemocytes) and reproduction conditions coupled with release of toxic (oocytes and sperms). byproducts, thus damaging the envi- This work was undertaken to evaluate ronment. An alternative approach is the role of mycobiosynthesis silver na- their production by the process of my- noparticles against B. alexandrina cosynthesis, using different of snails and its effects on some snails’ fungi which act as nanobiofactories, hematological and biological parame- since they produce and secrete enzymes ters. Also, the study aims to test the which help in reduction of metal ions to possibility of using these compounds as nanoparticles. They are further easy to a biological control for schistosomiasis culture, maintain and due to considera- vector snails. ble biomass favor the large scale pro- Materials and Methods duction of a wide variety of metal na- Laboratory bred adult Biomphalaria noparticles including those of silver, alexandrina snails (8-10 mm) were ob- gold, iron, cadmium, selenium and plat- tained from the Medical Malacology inum. They have been found to possess Laboratory, Theodor Bilharz Research monodispersity, stability and other fa- Institute (TBRI), Giza, Egypt. vorable properties which have led to Seven fungal isolates: Penicillium au- their wide usage and applications. rantiogresium (IMI 89372); P. roque- However, biological methods, which forti (IMI 285518); Aspergillus niger make use of fungi, bacteria and plants, (NRRL, 595); Verticillium chlamydo- are more ecofriendly and are being ex- sporium var. (CBS 600.88); Tricho- plored as alternatives to conventional derma viride (RCMB 004001); T. techniques which invariably involve longibranchiatum (RCMB 004006) and hazardous waste generation (Das & Paecilomyces variotii (RCMB 004101) Thiagarajan, 2012). were kindly provided from the culture The fungus Aspergillus fumigatus collection unit of the Regional Center (Sastry et al., 2003) fungus resulted in for Mycology and Biotechnology the rapid synthesis of silver nanoparti- (RCMB) Al-Azhar University, Cairo- cles and the particles formed were Egypt. All fungal isolates were sub- monodisperse which can be used in bac- cultured and maintained on Sabouraud’s terial applications. Aspergillus flavus Glucose agar (SGA) medium contain- (Mahendra et al., 2009) also resulted in ing (g/l); glucose-20; peptone-10; agar- the accumulation of silver nanoparticles 20 and distilled water,1000ml, pH of on the surface of its cell wall when in- medium was adjust to 5.4±0.2 at cubated with silver nitrate solution. 25±2°C. Circulating hemocytes represent the Previously, all fungal isolates were sub- primary mediator of cellular defense jected for the extracellular biosynthesis reactions in molluscs. Hemocyte mor- of AgNPs, studying their optimal condi- phology is continuously being given tion and characterized using UV-Visible importance and several studies have Spectral Analysis, FT-IR, EDX, XRD been conducted on snail species (Noda and TEM analyses in an earlier work and Loker, 1989; Barraco et al, 1993; (Helmy and Mekawey, 2013). Kambale and Potdar, 2010; Di et al, The seven fungal AgNPs compounds 2011; Cavalcanti et al, 2012). Schisto- were investigated against B. alexan- some vector Snail’ elimination could be drina snails by the exposure of 10 snails possible through applying safe biocon- to each compound for 24 h at 100 ppm trol agents affecting its immunity (Cir- concentration. Then they maintained in

628 dechlorinated water 24 h for recovery (Babson, 1965) enzymes in the hemo- and the mortality rate for snails was lymph of B. alexandrina snails were calculated. The promising compounds detected. Tested biochemical parame- were chosen to complete the experi- ters were evaluated spectrophotometri- mental study. cally by using kits from Quimica Clini- For molluscicidal screening, a stock ca Aplicada S. A. (QCA) Ltd., Span. solution of the tested compounds (1000 The concentrations of progesterone, ppm) was prepared on the basis of W/V Testosterone and Estradiol hormones using dechlorinated tap water (pH 7.0– were also determined by gas-liquid 7.5). A series of concentrations that chromatography according the method would permit the computation of LC50 of La atikainen et al. (1973). and LC90 values were prepared from the For total and differential counting, compound (WHO, 1965). Three repli- monolayer of hemocytes were stained cates were used, each of 10 snails (6–8 with Giemsa stain for 20 minutes, ac- mm)/L of each concentration. Exposure cording to the methods of Abdul-Salam and recovery periods were 24 h each at and Michelson (1983), examined and 25±1°C. For each test, three replicates counted by light microscopy and photo- of control snails were maintained under graphed by Toup View Camera. Anoth- the same experimental conditions. The er sample for each group was with- effectiveness for each compound has drawn from the region of the hermaph- been expressed in terms of LC50 and rodite gland (after gentle crushing) and LC90. stained as previously mentioned for the B. alexandrina snails exposed 24 hours examination of ova and sperms. to the sublethal concentrations (LC10 Statistical analysis was performed by and LC25) of the promising fungal silver the SPSS computer program (version 20 nanoparticles compounds in all experi- for windows). ments. Results For hemolymph collection, the snails The silver nanoparticles (AgNPs) of were dried by a paper towel and placed Penicillium aurantiogresium (IMI on their left side (i.e. spiral clockwise) 89372); P. roqueforti (IMI 285518); on the bottom of a dry Petri dish. A Aspergillus niger (NRRL 595); Verticil- small portion of the shell situated di- lium chlamydosporium var. (CBS rectly above the heart was removed and 600.88); Trichoderma viride (RCMB a syringe was inserted into the heart to 004001); T. longibranchiatum (RCMB collect the hemolymph as described by 004006) and Paecilomyces variotii Negm et al. (1995). (RCMB 004101) were investigated for The levels of total protein (Domas, their molluscicidal activity against 1975), albumin (Gustafsson, 1976), vector snails (Bi- glucose (Trinder, 1969) and lipids con- omphalaria alexandrina). Table (1) re- centrations (Knight et al, 1972) were vealed that the AgNPs of two fungi on- determined in the collected hemolymph. ly (Paecilomyces variotii and Aspergil- Also, the activities of transaminases lus niger) have molluscicidal activity Aspartate (AST) and alanine aminotre- (85 and 95% mortality) at concentration ansferase (ALT) (Reitman and Frankel, of 100 ppm when exposed 24 hours and 1957; White et al, 1970), the phospha- recovered for another day. tases; acid phosphatase; AcP (Moss, 1984) and alkaline phosphatase; AkP

629 Table 1: Molluscicidal effect of fungal AgNPs against B. alexandrina after exposure to 100 ppm for 24 hours. Fungal AgNPs Snails' mortality % Penicillium aurantiogresium 0 Penicillium roqueforti 0 Verticillium chlamydosporium 0 Trichoderma viride 0 Trichoderma longibranchiatum 0 Paecilomyces variotii 85 Aspergillus niger 95 Molluscicidal activity of silver nano- other 24 h recovery (Tab. 2). Data ob- particles of the two promising fungal tained indicated that A. niger AgNPs is AgNPs (Paecilomyces variotii and As- more toxic to B. alexandrina than P. pergillus niger) against B. alexandrina variotii AgNPs. snails after 24 hours of exposure and Table 2: Molluscicidial activity of Paecilomyces variotii and Aspergillus niger AgNPs against adult B. alexandrina (24 hours exposure). LC10 LC25 LC50 LC90 SLOPE Paecilomyces variotii AgNPs 65.5 74.7 85.0 104.5 1.21 Aspergillus niger AgNPs 32.6 47.9 64.8 97.0 1.54 Regarding biochemical analysis (Tab. the short period of exposure to both P. 4) the exposure to the tested compounds variotii and A. niger AgNPs. Also, the led to disturbance in the levels of the alkaline phosphatase enzyme increased steroid hormones in B. alexandrina he- significantly while the acid phosphatase molymph. The progesterone levels de- showed no significant differences com- creased significantly, while the levels of pared to control. testosterone and estradiol increased. A. Also, the concentration of the total pro- niger AgNPs did not affect the testos- tein, glucose, albumin and total lipids terone hormone. The liver enzymes are not affected significantly (p>0.05) (ALT &AST) increased significantly by when compared to the control group. Table 3: Effect of Paecilomyces variotii and Aspergillus niger AgNPs sublethal concentrations on some biochemical parameters and steroid hormones of B. alexandrina after 24 h exposure. P. variotii AgNPs A. niger AgNPs Control LC LC LC LC Test 10 25 10 25 (65.5 ppm) (74.7 ppm) (32.6 ppm) (47.9 ppm) Mean SD Mean SD Mean SD Mean SD Mean SD Progesterone (Nmol/L) 0.51 0.05 0.37* 0.03 0.39 * 0.04 0.36 * 0.03 0.31 ** 0.04 Testosterone (Nmol/L) 0.46 0.04 1.55*** 0.07 1.33*** 0.06 0.41ns 0.02 0.52ns 0.05 Estradiol (Pg/ml) 23.9 3.50 33.1* 3.81 36.1** 2.67 41.6** 4.11 42.8** 3.08 ALT (U/ml) 179 9.01 183ns 6.23 423*** 11.08 201* 9.88 280*** 4.46 AST (U/ml) 80 5.31 214*** 7.67 235*** 8.09 212*** 6.33 747*** 13.04 Alkaline phosphatase(U/L) 84 7.02 108* 9.11 181*** 5.32 317*** 7.91 410*** 9.54 Acid phosphatase (U/ml) 0.08 0.02 0.06ns 0.01 0.05 0.03 0.07 ns 0.02 0.06 ns 0.04 Total protein (mg/100 ml) 2.84 0.82 3.1ns 0.67 3.4ns 0.35 3.45 ns 0.79 2.7 ns 0.56 Glucose (mg/100 ml) 31.9 4.51 30.6ns 6.11 35.3ns 5.53 33.6ns 8.01 35.8 ns 7.09 Albumin (g/l) 1.8 0.80 1.9ns 0.09 1.8ns 0.51 1.7ns 0.46 2.0ns 0.08 Total lipids (mg/100 ml) 352.6 15.38 375.6ns 13.44 352.1 9.78 321.1 14.11 322.7ns 14.47 *, ** & *** = significantly different from control at p<0.05, p<0.01 & p<0.001, ns= no significant difference (p>0.05). In hematological study, total count of crease by 17.4-47.8% in the snails ex- the hemocytes showed significantly in- posed to both A. niger AgNPs and P.

630 variotii AgNPs sublethal concentrations exposed to LC25 from A. niger and P. 24 h compared to control ones (Tab. 3). variotii AgNPs, respectively. On the Differential count indicated a signifi- other hand, the hyalinocytes increased cant reduction in the large granulocytes significantly in treated groups than the by 44.7% and 28.9% (p<0.001) in snails untreated control. Table 4: Total and differential count of B. alexandrina hemocytes after exposure to Paecilomyces variotii and Aspergillus niger AgNPs for 24 hours. Total Count Differential count % AgNPs compound Mean SD LG SG Hy Control 2300 101.4 76 13 11 P. variotii (LC10) 2700 * 165.7 62 * 10 ns 18 ns P. variotii (LC25) 2900 * 202.8 42 *** 8 ns 50 *** A. niger (LC10) 3200 ** 300.0 51 *** 6 ns 43 *** A. niger (LC25) 3400 ** 265.7 54 *** 6 ns 40 *** LG = large granulocyte, SG = small granulocyte, Hy = Hyalinocyte, *, ** & *** = significantly dif- ferent from control at p<0.05, p<0.01 & p<0.001, ns= no significant difference (p>0.05)

The light microscopy photographs of B. toplasm (Fig. 1, G) and the Hy cells ap- alexandrina hemocytes (Fig. 1, Con- peared with cytoplasmic vacuoles (Fig. trol) showed two morphologically dis- 1, H). tinct types of hemocytes, granulocytes The exposure to LC10 of A. niger and hyalinocytes. Granulocytes are dis- AgNPs for 24 h showed enlarged divid- tinguished to two types, large and small ing LG (Fig. 1, I), phagocytosis (Fig. 1, granulocytes with a low nucleus to cy- J) and lysed Hy (Fig. 1, N). When B. toplasm ratio. Large granulocyte cells alexandrina exposed to LC25 of A. niger (LG) are variable in size with small nu- AgNPs, the hemocytes’ morphology cleus and high cytoplasmic content due showed obvious abnormalities. Granu- to the presence of refractile granules. locytes and hyalinocytes showed cell Small granulocytes (SG) are intermedi- apoptosis with degenerated cytoplasm ate cells with several micron sized with released granules and shrinked nu- granules and distinguished from LG by cleus (Fig. 1, L- N). the central location of the nucleus and a The impact of the tested compounds relatively constant size. The hyalinocyte on the morphology of B. alexandrina cells (Hy) are characterized by small oocytes and sperms was illustrated in size and they are circular shape with figures (2 & 3). Figure (2, A) showed clear dense cell membrane, large nucle- the normal mature oocyte and sperm in us and basophilic cytoplasm with a high control group. B. alexandrina treated nucleus to cytoplasm ratio. with LC10 of P. variotii AgNPs showed B. alexandrina snails exposed to LC10 terminal and divided nuclei (Fig 2, B & of P. variotii AgNPs for 24 h showed C), sperm head separated from its tail some abnormalities in LG cells; eccen- (Fig 2, D) and in Fig (2, E) the sperm tric nucleus (Fig. 1, A) nucleus division showed pin head and thickened tail. The (Fig. 1, B) and the hyalinocytes ap- exposure to LC25 led to light degenera- peared with vacuolated nucleus. When tion of the oocyte contents even in the the concentration increased to LC25, the oocyte division (Fig 2, F & G), the LG cells showed abnormal dividing sperms showed swallowed head and with highly vacuolated cytoplasm (Fig. thin tail (Fig 2, H) and other sperms 1, D), cell wall invagination (Fig. 1, E) showed abnormal swallowed head with pseudopodia (Fig. 1, F), vaculated cy- severely nodded tail (Fig 2, I).

631 The exposure to A. niger AgNPs level of progesterone in Fenitrothion showed strong toxic effects on the oo- treated snails. In addition, the levels of cytes and sperms of snails. At LC10 ex- testosterone hormone increased signifi- posure, the photographs showed dead cantly in snails treated with P. variotii aggregated ova (Fig. 3, A), sperms ap- AgNPs while no significant changes peared in necklace shape (Fig. 3, B) and were observed at the exposure to A. ni- other showed swallowing of sperms ger AgNPs. This agreed with Rizk et al. heads. Increasing the concentration to (2012) who found disturbance in the LC25 led to the appearance of swal- level of steroid hormones in B. alexan- lowed, vacuolated and degenerated ova drina treated with Haplophyllum tuber- (Fig. 3, D), also other oocytes appeared culatum. with large fat vacuoles which led to de- Also, the concentration of estradiol generation of its contents (Fig. 3, E). in hormone increased significantly in this addition, the photographs showed investigation with the increase of the atretic oocyte surrounded by dead two fungal AgNPs concentration. The sperms with wrinkled tails (Fig. 3, F) high concentration of estradiol in treat- and hyperplasia and necrotic sperms ed snails may be related to increasing heads led to overlapping of tails (Fig. 3, rates of proliferation and protein syn- G). thesis that in turn leads to increase in Discussion total protein content as documented in The present study results revealed that the present results. On the other hand, the silver nanoparticles of two fungi Mohamed and Aly (2014) found de- (Paecilomyces variotii and Aspergillus crease in estradiol hormone and total niger) from seven proved to have mol- protein concentrations in Fenitrothion luscicidal activity against B. alexan- treated snails. The significant elevation drina (at concentration of 100 ppm) of in ALT, AST and AkP in treated snails 85% and 95%, respectively. Ghoneimy is in agreement with Al-Sayed et al. et al. (2014) recorded toxic five fungal (2014) who found that AkP and AcP strains extracts (Aspergillus sclerotium; were significantly increased by 38.5 and Chaetomium globosum; Penicillium ca- 48.4% in the hemolymph of B. alexan- nescens; Penicillium griseofulvum and drina exposed to LC25 of the methanol Trichoderma longibrachiatum) from extract of E. globulus and M. stypheli- thirty against B. alexandrina and Buli- oides, respectively. In addition, nus truncatus snails. These results are Ghoneimy et al. (2014) found that all also, in parallel with that of El- the liver enzymes (AST, ALT, AcP and Hommossany and El-Sherbibni (2011) AkP) in the hemolymph of the exposed who recorded lethal effect of the photo- snails were elevated significantly by the sensitizer Hematoporphyrin coated gold exposure to LC25 of the Penicillium ca- Nanoparticles (Hpd coated GNPs) nescens bioactive compound for 4 against B. alexandrina. weeks. The present results reported signifi- The hemocytes total count of snails cance decrease in the levels of proges- exposed to sublethal concentrations of terone in snails treated with the two P. variotii and A. niger AgNPs showed fungal AgNPs, which may lead to poor significant increase compared to control reproductive efficiency as a manage- group. The elevation of total hemocytes ment for control snails’ strategy. This is the most commonly observed re- agrees with the results of Mohamed and sponse in different molluscan species Aly (2014) who found significant low exposed to different stressors. Russo

632 and Lagadic (2004) stated that, the in- same results when using different plant crease in hemocyte density might be molluscicides on hemocyte of B. alex- seen as a way for the snails to maintain andrina and B. truncatus. These ab- a constant level of global phagocytic normalities recorded in the hemocytes activity, which was inhibited as a result were also confirmed with the findings of treatment. In agreement with this re- of Sharaf El-Din (2003), Souza and sult, Mohamed (2011), found that expo- Andrade (2006), Kamel et al. (2006) sure of B. alexandrina snails to suble- and Mossallam et al. (2013). thal concentration of Roundup herbicide Severe abnormalities also were de- led to significant increase in total he- tected in B. alexandrina oocytes and mocytes counts during 7 days of expo- sperms due to the toxic effect of P. var- sure. Consequently, the spreading he- iotii and A. niger AgNPs in the present mocytes increased during the whole ex- work. The photographs showed abnor- perimental period. mal nuclear division, degeneration and The present data recorded significant large fat vacuoles in the cytoplasm and reduction in the number of granulocytes swallowed atretic oocytes. Also, the by 19.1- 43.8% and increase in the photographs showed dead sperm head number of hyalinocytes by 63.6-354.5% separated from its tail, other sperms in treated experimental groups. In showed abnormal swallowed head with agreement with these results, Gawish et severely nodded tail, dead sperms with al. (2008) recorded a reduction by wrinkled tails, hyperplasia and necrotic 34.9% in granulocytes of Bulinus trun- sperm heads led to overlapping of tails. catus continuously exposed to LC10 of These abnormalities may be due to the the Callistemon citrinus extract for one inhibiting action of the fungal AgNPs week, while amoebocytes and hyalino- on oogenesis and spermatogenesis in cytes were increased by 54.4% and the ovotestestis gland. These results in 21.7%, respectively. Mossallam et al. agreement with Abdel-Hamid et al. (2013) found a decrease in the number (2014) who recorded ova and sperm of B. alexandrina hemocytes when ex- abnormalities as a result of B. alexan- posed continuously to LC25 Artemether. drina and B. truncatus exposure to sub- Similar results were found by Sharaf lethal concentrations of P. canescens El-Din (2003), Souza and Andrade bioactive compound. Also, Bakry et al. (2006) and Kamel et al. (2006, 2007). (2002) found that, B. alexandrina snails The light microscopy photographs of exposed to fungicide (Isoprothiolane) untreated B. alexandrina hemolymph laid highly significant number of ab- showed two morphologically distinct normal egg masses. Pagano et al. types of hemocytes; granulocytes and (2001) showed significant toxicity of hyalinocytes. This was agreed with R6 fungicide on sea urchins resulting in Sminia and Van der Knaap (1987), Lie embryo developmental defects, trans- et al. (1987), Barraco et al. (1993) and missible damage from sperm to the off- Serrano et al. (2002). spring and cytogenetic abnormalities. The exposure of B. alexandrina to Conclusion LC25 of both P. variotii and A. niger The applying the biosynthesized com- AgNPs showed apoptotic hemolymph pounds which led to destruction of cells, fragmented, vacuolated and de- blood cells (the immune system), ova generated cytoplasm, shrinked nucleus and sperms (the reproductive system) of and phagocytosis. Kamel et al. (2007) snails is an important effective step to and Gawish et al. (2008) reported the control schistosomiasis.

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635 Explanation of figures Fig. 1: Light microscopy photographs of Biomphalaria alexandrina hemocytes showing two types of hematocytes in the control; granulocytes (large [LG] and small [SG]) and hyalinocytes (Hy). Photgraphs showing hemocytes of B. alexandrina exposed for 24 h to LC10 of Paecilomyces variotii AgNPs (A-C), LC25 of P. variotii AgNPs (D-H), LC10 of Aspergillus niger AgNPs (I-K) and LC25 of A. niger AgNPs (L-N) (1000X). Fig. 2: Light microscopy photographs of Biomphalaria alexandrina oocytes and sperms, control showing normal mature ovum and sperm (A), oocytes and sperms of B. alexandrina exposed for 24 h to LC10 of Paecilomyces variotii AgNPs (B-E) and LC25 of P. variotii AgNPs (F-I) (1000X). Fig. 3: Light microscopy photographs of oocytes and sperms of Biomphalaria alexandrina exposed for 24 h to LC10 of Aspergillus niger AgNPs (A-C) and LC25 of A. niger AgNPs (D-G) (1000X).

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