Saprolegnia is an oomycete that has long been considered as a separate class within the kingdom Fungi. However, sequence DNA comparisons indicate that they are closer to brown algae, with a worldwide distribution [1,2]. The growth sign of the disease typically is manifested as a relativity superficial, cotton-wool like, white growth of mycelia on the fish skin specially around the head, dorsal and caudal fins, gills, in muscular layer and internal organs [3-5]. Even though Saprolegnia sp. can affect amphibians, crustaceans and many fish species it frequently affects salmonids, tilapia species and fish ova [6-7]. Several species of Saprolegnia have been found to be responsible for fish infections called saprolegniasis, which cause several damages in natural ecosystems as well as important economic losses for the aquaculture industry [2,8]. Some outbreaking factors for saprolegniasis can be seed stage, mechanical damage, stress, immune compromise, reduces water quality, poor hygiene and overpopulation [9].

Research for taxonomic identification of Saprolegnia sp. uses classical morphological criteria based on reproductive structures i.e. antheridia, oogonia and oospores [9-11]. These criteria set with variations in esterase isoenzyme patterns [12] and differences in radial growth rate [13] allow classifying isolated Saprolegnia sp. strains in different subgroups [14]. On the other hand, recent research shows that the identification of Saprolegnia species using traditional taxonomic criteria and keys on parasitic isolates, at best, has proven problematic [15]. Some isolated Saprolegnia strains did not produce reproductive structures and were classified as Saprolegnia parasitica [16]. Due to the uncertain classification of Saprolegnia sp. by morphological criteria and the great variability in the group, recently, molecular taxonomic tools were applied aiming to a more specific classification [1,17,18].

On the other hand, the study of these microorganisms is too difficult because of its complicated isolation and culture in laboratory, since research it can only be done in pure cultures [19]. Once pure culture is obtained it is possible to study the components of the cell wall. Saprolegnia species have different types of molecular component on it Cell wall as: 1-3 glucans, 1-6 glucans and cellulose whereas chitin, which is a major cell wall component of fungi, has been shown to occur in very small amounts in the walls of oomycetes. These cell wall molecules can be recognized by the immune system of fishes and other organisms [20,21]. There is little understanding of the fundamental molecular mechanism involved in the interaction between pathogen and host thus, the knowledge of these molecules could help to better understand the pathogenicity and the immune response during the infection of Saprolegnia sp.

In the present study we isolated a strain of Saprolegnia sp. obtaining pure culture with a cotton-wool mycelia and reproductive structure as antheridia and zoospores in laboratory conditions and, in parallel we designed a kit made up of immune component of the insect D. coccus homopteran that can detect the presence of Saprolegnia sp. isolated strain in mycelia and spore stages.

Reagents

Reagents of the highest purity grade available were obtained from Sigma Chemical Company (St. Louis MO) and BD Bioxon, Becton Dickinson of Mexico.

Fungus isolation

The strain of Saprolegnia sp. was obtained from skin lesions on tail and ventral part of the body from dying goldfish (Carassius auratus) and water samples of the tanks containing diseased fishes. The samples were inoculated and maintained at 4°C on potato dextrose agar (PDA) with an ampicilin solution at 0.4 mg/ml^-1 to avoid bacteria, and routinely inoculated (every 48 hr) on a new PDA Petri dish with ampicillin, until there were no fungi or bacterial growth, in order to obtain pure strain.

Insects

D. coccus female insects were collected from cladodia of Opuntia ficus-indica maintained in greenhouse at 24°C-26°C. Under these conditions, the life cycle lasted approximately 3 months for the wingless adult females, which are attached of the cactus by their proboscis. Fifteen female insects were collected when they reached a size of about 6.0 mm long.

Hemolymph collection

Adult female insects were carefully scraped from the prick pear pads and perfused through with 50 μl of saline solution (NaCl 0.9%). Hemolymph (HL) was transferred to a polypropylene microtube and centrifuged at low speed (600 g) to remove debris. Supernatant was recovered and kept on ice until use. Granulocytes were recovered from the supernatants and their integrity was analyzed under phase contrast optics (Nikon E-600 microscope). The hemolymph protein concentration was determined by the Lowry method [23].

Strain characterization

From the pure strain of Saprolegnia sp. obtained with the continues growth on PDA-ampicilin medium, reproductive structures of Saprolegnia sp. were promoted by growing the isolated strain on common fly cadavers (Musca domesctica), due to its rich chitin exoskeleton that is a source of nutrients for Saprolegnia development, and Sabouraud dextrose agar for 10 days at 20ºC. The cultures were observed under an optical microscope (Carl Zeiss Axiostar Plus- Germany) to check the presence of sexual and asexual structures.

Hemolymph activation assays

Zymosan (commercial purified component of fungus cell wall) [15] stock solution (100 mg glucose equivalents/ml) was prepared as described by Lanz-Mendoza et al. [23] 50 μl of hemolymph prepared as described above (6.4 mg/ml of protein) were mixed with zymosan (50 μg/ml) and spore suspension (1:2) of the Saprolegnia sp. isolate on sterile water, volume was completed to 500 μl with saline solution 0.9%. Samples were incubated at 37°C for 15 min without shaking. After centrifugation at 1,000 xg, supernatants were transferred to new tubes to be readed by spectrophotometry. Carminic acid consumption was measured in a spectrophotometer (Beckman DU 640) at 495 nm. The results represent the average of three different experiments done by duplicate.

Dosage-response assays

Spore suspension (1:2) was prepared in saline solution (0.9% NaCl) in consecutive volumes between 5 μl to 300 μl, mixing 50 μl of HL. The samples were incubated 30 min at 37ºC. Then centrifugated at 1,000 xg to detect melanin clot and consumption of carminic acid described as above.

Degranulation and melanin production on D. coccus granulocytes

To determine the production of melanin and degranulation in D. coccus granulocytes, HL was collected as described above and centrifuged at 1,000 xg for 5 min. The pellet containing degranulocite was collected and re-suspended in 250 μl of Schneider’s insect medium. Fifty microliters of the cell suspension were placed on cover slips. Cells were allowed to attach for 10 min and then incubated with 5 μl of zymosan (50 μg glucose equivalents/ml) or 5 ml of spore suspension. Control samples without zymosan were also included. Cells were analyzed using an inverted microscope (LEICA-DMLS). Photomicrographs were obtained with a PENTAX 2 X-M camera.

Morphology and physiology

The main morphological characteristics of the Saprolegnia sp. strains are presented in Figure 1. The isolated strain demonstrated aseptate hyphae (Figure 1A) and zoospore release representative of Saprolegnia sp. All asexual isolates at 20-22ºC were classified as Saprolegnia sp. due to the presence of the complete asexual cycle with zoosporangium (Figure 1A), primary (Figure 1B) and secondary zoospores (Figure 1C), encysted secondary zoospore showing sign of a small germ tube (Figure 1D) and germling secondary cyst (Figure 1E) like the ones reported by Burr and Beakes for Saprolegnia parasitica. The strains maintained in PDA broth produce bundles of new hyphae at 20-22ºC for a period of 48 hr, and hyphae samples were incubated in sterilized water between 20-22ºC for 48 hr and obtain typical asexual structures.

Figure 1: Seedlings at 2 weeks after sowing: showing period of insect infestation.

Fungal effects on granulocytes of D. coccus

Zoospores and zymosan in the concentration mention above, as control, were added in granulocytes cultures (Figure 2B1) and incubated at 36ºC in Schneider’s medium for 15 min, in order to know the response of the granulocytes with the presence of Saprolegnia sp. Under these conditions granulocytes degranulate (Figure 2B2) and oxidate carminic acid as a response of the interaction with fungus elicitors (Figure 2C1 and C2).

Figure 2: Sequence response of granulocytes to Saprolegnia sp. elicitors.

D. coccus hemolymph activation with Saprolegnia sp.

As described above HL was activated with zoospores of Saprolegnia sp. and produced a lowering the absorbance at 495 nm, maximum absorbance of carminic acid dye (Figure 3), indicating that carmine acid dye was consumed during the reaction and a fibrilar aggregate precipitate (Figure 2C2). The hemolyph was activated after 15 min of exposition to both zymozan and Saprolegnia sp. at 36ºC. LPS (50 μg/ml) neither induce red pigment consumption nor black fibrillar aggregation (data not shown).

Figure 3: Red pigment of D. coccus hemolymph is consumed during clotting reaction. Hemolymph of D. coccus was incubated alone or in the presence of fungus elicitors in the range 400 to 700 nm and was measure 15 min incubation time. Rhombus ◊, HL alone; Square ■, HL with zymosan 50μg/ml; Triangle ▲, HL incubated with Saprolegnia sp.

Sensibility and dosage-response relation of D. coccus hemolymph with Saprolegnia sp. zoospores

The 2 ml hemolymph collected was incubated with Saprolegnia sp. zoospore suspension, with different concentrations, showing a dosis response to oomycete elicitors, and a black precipitate (Figure 2C) with consumption of carminic acid dye present in HL. 300 μg of HL protein was sensitive to the presence of 5 zoospores of Saprolegnia sp (Figure 4), with an absorbance of 0.503, to 282 zoospores with an absorbance of 0.103. The absorbance diminution represents about a 65% respect to the control with an absorbance of 0.202 Figure 4.

Figure 4: Relation between the concentration of Saprolegnia sp. zoospores and carminic acid dye consumption, express in absorbance. Independent tubes with consecutive volumes of Saprolegnia sp. zoospores suspension incubated with HL, obtaining an absorbance of 0.053 with a minimum of 5 zoospores and 0.103 with a maximum of 282 zoospores.

Taxonomical classification of organisms from the genus Saprolegnia has been debated for years, first because of its similarity to brown algae better than to fungus and then because isolated species such as S. parasitica S. anomalies, S. anisospora, S. asterophora, S. australis, S. bulbosa, S. diclina, between others, of the same taxonomic grouping show considerable variation in pathogenicity, production of reproductive structure in laboratory and phenotype. The strain isolated in our experiments was classified as Saprolegnia sp. by morphological criteria due to the presence of cenocitic hyphae, absence of oogonia (sexual stage) and the presence of all the asexual stages such as: zoosporangium, zoospores and cyst in laboratory conditions as reported for this genus by Burr and Beakes, Mortada et al., Gunnar and Cerenius [24-26].

Another typical cell wall component of this genus are β 1-3 glucans, micolaminarins, cellulose polymers and few chitin. Hernandez- Hernandez et. al. reported that components of the HL of D. coccus have the capacity to recognize molecules such as: β 1-3 glucans, zymosan and laminarin with melanin production. In this protocol when HL from D. coccus was incubated with Saprolegnia sp. strain, there was a consumption of carminic acid dye with the consecutive production of melanin as a result of the recognition of Saprolegnia sp. cell wall components [27]. The same reaction was observed when HL granulocytes were incubated with Saprolegnia sp., with a dreganulating reaction of carminic vesicles, located in the cytoplasm of the cell [28]; and melanin formation, as reported in Hernandez- Hernandez et al., when incubating HL with components purified of fungus wall cell, concluding that HL recognizes Saprolegnia sp. cell wall components [27]. This reaction can be part of immune response of D. coccus against natural pathogens as described in other arthropods like Bombix mori and Manduca sexta [3].

In order to apply this knowledge for resolving aquaculture problems, as the prevention of saprolegniasis, there is a possibility to create a quantitative probe, with the sensibility of HL against the presence of at minimum 5 zoospores of Saprolegnia sp. As being a motile stage, increases the possibility to be found in water samples. The reaction was made with define quantities of zoospores, observing that the diminution of optical density at 495 nm was inversely proportional to the quantity of zoospores using constantly 50 μl of HL (6.4 μg/ ml protein). There is another kind of “commercial kit” which use a similar response described here and is based in the reaction of lysate of amebocytes of Limulus. In this case, the presence of endotoxin in solution elicits a reaction of coagulation. This principle is used in order to detect the presence of endotoxin from enterobacteria [29].

In summary the results showed in this work increase the knowledge of D. coccus immunity, and in this case the opportunity to apply a colorimetric method that can detect Saprolegnia sp., opening the possibility to develop biotechnological tools for aquaculture problems, generated from the study insects immune response. This results suggest the participation of molecular receptors, signal pathways and the presence of enzymes cascade that are activated by the recognition of oomycete elicitors which opens new research ways for fish disease detection in order to an efficient control of infections.

We thank the personnel of the Molecular Entomology laboratory from CINVESTAV-IPN and laboratory assistance of the School of Biology-Simon Bolivar University. Also special thanks to the biologist Rosario Vazquez Bravo, M.Sc. Alfonso Montañes Arce for technical assistance and Ing. Ignacio del Rio y Dueñas (“Tlapanocheztli” Foundation) for providing biological material. This project was financially support from CONACYT project 61326.