A novel disease of crayfish Procambarus clarkii appeared in the summer of 2004 in freshwater aquaculture in Jiangsu province of China. Light and transmission electron microscopy (TEM), molecular biological methods and in vitro culture were used to identify the pathogen. The agent was unique in having a helical morphology and rotary motility as observed by phase-contrast light microscopy and was found in haemolymph, muscles, nerves and connective tissues by smear method and TEM. Ultra-thin sections under TEM revealed the wall-free membrane of the microbe. The agent could pass through membrane filters with pores 220 nm in diameter and was cultivated in vitro in M1D medium. 16S rDNA of the crayfish pathogen was amplified by PCR using primers specific for Spiroplasma-specific 16S rDNA. The resultant 271 bp PCR product showed 99% identity with Spiroplasma mirum 16S rDNA, having a close relationship with the spiroplasma from the Chinese mitten crab Eriocheir sinensis. This is the second time a spiroplasma has been found in a freshwater crustacean. The 271 bp PCR product was also amplified from the bottom mud in the ponds associated with the disease. The PCR molecular method is an effective way to detect spiroplasma in freshwater environment. The results from this study are significant in expanding the host range of spiroplasma and freshwater ecology.
Red swamp crayfish is a freshwater crustacean of the genera Procambarus clarkii, resembling a lobster but considerably smaller. This species, originally from America, was introduced into Japan in 1918 as bait for breeding bullfrogs and multiplied and proliferated abundantly in later years. The species was brought to Nanjing, China during the Second World War from Japan. Since the 90's crayfish have been farmed extensively as food and have become an important aquaculture product in the southeast of China, especially in Jiangsu Province. In the summer of 2004, a novel weakening disease leading to death was observed in the crayfish. The diseased crayfish were found in an aquaculture pond with the Chinese mitten crab, Eriocheir sinensis, at the same time that a serious disease of the crab, tremor disease (TD), occurred in the same pond.
TD of Chinese mitten crab has become an economically important disease of the crab in recent years . The causal agent, tremor disease agent (TDA), was first thought to be a rickettsia-like organism (RLO) by epidemiologic and pathological studies [2,3] but later was proven to be a spiroplasma by 16S DNA sequence analysis and motility detection by dark field light microscopy [4,5]. This is the first time a spiroplasma has been detected and characterized in a crustacean. Red swamp crayfish, which are also a decapod crustacean, are close relative of the mitten crab. It is possible that there maybe a relationship between TD of the crab and the disease of the crayfish. This paper describes the studies on the disease of the crayfish and the relationships with TD and the aquatic environment by light and electron microscopy, molecular biological methods and in vitro culture.
2Materials and methods
2.1Isolation and cultivation in M1D medium
The diseased crayfish, as indicated by rapid testing for hemocytes from living crab and crayfish , were first disinfected with 75% alcohol. A needle was inserted into the diseased crayfish through the joint between two middorsal arthromeres and 0.5 ml of haemolymph was obtained with a 2 ml syringe. The haemolymph was mixed with an equal volume of sterile PBS. The homogenate was passed through a filter with 220 nm pores and collected in tubes. The isolate was used for in vitro culture in M1D broth [7–9]. Tubes containing 1.5 ml of medium were inoculated with 0.05 ml of the fluid isolate, incubated at 30°C and observed daily. Colour change was noted to determine if growth was established in each of the tubes. Upon acidification of the cultures, light microscopy was used to examine the media for the presence of organisms and to determine their morphology. Subculture passages were made to ensure that growth was sustained by the medium and at least 10 additional passages were carried out. Ten diseased and ten healthy (as control) crayfish were used for isolation and cultivation.
Water and bottom mud in three different aquaculture ponds associated with the disease were collected from five positions of each pond (one sample from each corner and one at the center of the pond) and filtered with qualitative filter papers first and then with a filter with 220 nm pores, respectively. The filtrates were used for in vitro culture in M1D broth by the method mentioned above. As controls, samples from two aquaculture ponds without the disease were treated the same way.
One hundred and twenty crayfish taken from market or aquafarms were examined initially by simple and fast light microscope detection technology . Healthy crayfish (n= 96) were chosen for experimental infection and divided into four groups, eight crayfish in each group for the transmission studies. All treatments were in triplicates. In the first treatment, Medley culture, healthy and diseased crayfish were maintained together. Twenty-four healthy crayfish and the same number of diseased crayfish were maintained together. In the second treatment, the haemolymph inoculation trial, healthy crayfish were each inoculated with 0.1 ml of haemolymph from a diseased crayfish. The third treatment was the isolate inoculation trial, in which healthy crayfish were each inoculated with 0.1 ml of cultured isolate in M1D medium. In the fourth treatment, Control inoculation, healthy crayfish were inoculated with sterile PBS as a control for possible handling mortalities (Table 1). The inoculations were made with a 1 ml syringe through the joint between two middorsal arthromeres of every crayfish. Haemolymph smears from all of the crayfish were collected every five days by a modified Geimsa method for detection of the agent, as described before [2,6]. To fulfill Koch's postulate, the haemolymph was once again sampled from the moribund crayfish, filtered, added to M1D media and following 7 days growth was examined by light and electron microscopy for the presence of the agent.
Table 1. Groups for experimental infection
Days of symptoms
Days agent can be detected in haemolymph
Blood inoculation trial
Isolate inoculation trial
2.3Light and electron microscopy
A drop of haemolymph and isolates in M1D media from the healthy and naturally or experimentally infected crayfish was put on a glass slide and covered with a glass-cover slip. All samples were observed under a phase contrast light microscope (Nikon 50i). A digital camera was used for recording the pictures and the motility of the organism.
Haemolymph smears from every crayfish, including the healthy and the infected, were collected for Giemsa staining before dissecting. Impression smears of hearts, gills, ventral muscles, thoracic ganglion and gonads from diseased and healthy crayfish were also stained with Giemsa . The smears were fixed on film with 10% methanol, air-dried, then placed directly in a glass Coplin jar containing Giemsa stain diluted 1:10 with phosphate buffer (pH 6.8) for 45 min. All stained samples were observed using an Olympus BH-2 microscope.
Negative staining employed PTA-stained preparations with 2% sodium phosphotungstate (PTA; pH 7.0). Drops of the isolates (from infected crayfish, cultivation and healthy crayfish as control), fixed with 2.5% glutaraldehyde, were placed on formvar-coated copper grids for 1 min. After removal of excess fluid by blotting on filter paper, the wet residues were immediately covered with the stain for 30–40 s, and then withdrawn in the same fashion. The grid was air-dried before examination. In preparation for ultrathin sectioning, specimens including tissues from infected crayfish, healthy crayfish (as control) and isolates from M1D cultivation in broth were first fixed in 2.5% glutaraldehyde made up in phosphate buffer, and then transferred to 1% osmium tetroxide in the same buffer, followed by serial dehydration with acetone and embedding of specimens in Epon 812. Ultrathin sections with a thickness of 50–80 nm were made by Reichet–Jung ultramicrotome and double-stained with uranyl acetate and lead citrate. The sections were observed and photographed by a Hitachi 600–2A TEM.
2.4Extraction and amplification of Spiroplasma ribosomal DNA
DNA from the isolated agents in culture from disease crayfish was extracted by Chelex-100 resin (Bio-RAD, Biotechnology Grade) DNA extracting method. The agents, during exponential replication in M1D medium, were washed with PBS and the deposits were mixed with 200 μl 5% Chelex-100 resin. The mixture was incubated at 56°C for 20 min, followed by an incubation at 99°C for 8 min and then centrifuged for 4 min at 13000 × g (4°C). In addition to the agent from crayfish, the water and the bottom mud from the ponds, both associated and unassociated with disease, were also treated by the above method. TDA was used as a positive control whereas Escherichia coli, human leucocyte and sample without DNA template were used as negative controls. Two different diluted isolates (1 × 10−7 and 1 × 10−8) in M1D were made by serial dilution and used for detecting optimal concentration for PCR and for comparison with the concentration of the samples from the environment. DNA of the appendage muscles from disease crayfish was extracted by Tissues/Cell Genomic DNA Isolation Kit made by Huashun Biol. Inc. (Shanghai) according to the manufacturers instructions.
The template DNA was amplified by polymerase chain reaction (PCR) using Spiroplasma-specific 16S rDNA primers  including forward oligo (F28, 5′-CGCAGACGGTTTAGCAAGTTTGGG-3′) and reverse oligo (R5, 5′-AGCACCGAACTTAGTCCGACAC-3′). PCR reactions were carried out using PTC-100 thermal cycle with heated lid (MJ). The PCR reaction mix for each sample included 2.5 μl STR 10X Buff4Taq DNA Polymerase, and 14.85 μl ultra pure H2O to a final volume of 25 μl. The sample was denatured at 96°C for 2 min, and then underwent 30 cycles of 94°C (1 min), 65°C (1 min) and 72°C (1.5 min) followed by an extension cycle at 72°C (10 min) and preserved at 10°C. The PCR products were electrophoresed with Mini-PROTEAN 3 Electrophoresis Cell (BIO-RAD) on 2% agarose under the conditions of 85 V, 26 mA, 2 W. The electrophoresis image was photographed by a Nikon-4500 camera and the 16S rDNA fragments were sent to UG Inc. for DNA sequence determination (3700, Bigdye-Terminator). Sequences were compared to those in the GenBank database using the program BLASTN. The sequence has been assigned the accession number of the crayfish spiroplasma (GenBank accession no. AY927996).
3.1Isolation and cultivation in M1D medium
The pathogen from the diseased crayfish and bottom mud could pass through a membrane filter with average pore diameter of 220 nm and could be cultivated in vitro in M1D broth. The pathogen grew slowly at the beginning with a doubling time of 7 d during the 1–3 passages. The organisms increased more gradually to a peak of about 1 × 108 cells/mL by day 7 and grew quickly with an average doubling time of 2 d. The pathogen was not isolated nor cultivated from the water from both control and disease ponds. It should be noted that isolated pathogen was not found in all positions of the bottom mud and showed territorial distribution in 3 ponds by 1/5, 2/5, 1/5.
3.2Morphological characteristics of the pathogen
The organisms isolated from diseased crayfish and bottom mud exhibited a classic spiral/helical form, pleiomorphic form and spherical form (Fig. 1). The rotary motility could be seen under phase-contrast light microscope. The microbe's pleiomorphic shapes included pear-shaped cells, flask-shaped cells with terminal tip structures, filaments of various lengths, and helical filaments that could be detected clearly with transmission electron microscope (TEM) by negative staining (Fig. 3). Ultrastructural examination of the agent showed the lacked a cell wall (Fig. 4) and was 50–200 nm in diameter and 2–14 mm in length. When dividing, the agents were irregular in shape, resembling dumbbells, awls, or crescents (Fig. 1B–D).
The results of experimental infection are presented in Table 1. The crayfish inoculated with haemolymph from infected crayfish or the isolate in M1D medium showed weakness symptoms around 18–26 days after inoculation and were dead within 35–40 days after injection with a 92% or 96% infection rate, respectively. The infection rate was determined by examining both mortal crayfish and survivors by fast light microscope detection technology . Medley culture group showed an infection rate of 54%. The crayfish in control group were healthy and had no symptoms for more than 3 months.
In all experimentally infected crayfish the spiroplasma-like agents were located in haemolymph and the connective tissues of the gonads, the pereiopods, gut, hepatopancreas, nerves, heart and gills as determined by TEM, whereas in healthy crayfish, no similar agents were found. The impression smears from infected crayfish stained pink to bluish-purple with Giemsa stain and inclusions in the haemolymph could also be detected by phase-contrast microscopy. The results from the experimental infections completed Koch's postulate indicating the spiroplasma-like organism was the causative agent of crayfish weakness disease.
3.416S rDNA sequence analysis
PCR amplification of the 16S rDNA of the isolates from diseased infected crayfish and bottom mud in 7/15 position of 3 ponds (2/5, 3/5, 2/5) associated with the disease yielded products of 271 bp (Fig. 2). Negative results were obtained with E. coli, human leucocyte, the sample without DNA and the bottom mud of all positions in the two ponds (0/5, 0/5) without the disease. Two products (Lane 6 from crab, Lane 8 from crayfish) of 271 bp were sequenced (Fig. 2). Comparison to the GenBank data base confirmed the presence of spiroplasma-like DNA, which was 99% identical to S. mirum. A single nucleotide base, at position 809, was changed from an A to C (Table 2). The GenBank accession number for the agent sequenced from the crayfish is AY927996. Compared to the sequence of crab spiroplasma (GenBank accession no. AY920929) there was 99% identical.
Table 2. Sequence of PCR product from crayfish and crab cases compared to Spiroplasma mirum 16S rDNA sequence
These studies have shown by experimental infection (fulfilled Koch's postulate), light and transmission electron microscopy that a spiroplasma-like organism is the causing agent of crayfish weakness disease with systemic infection.
Molecular biological methods and in vitro culture showed that the agent isolated from the crayfish is a spiroplasma. The PCR amplified 16S rDNA has 99% identity with that of Spiroplasma mirum. This is the second freshwater crustacean host of spiroplasma. Although the crayfish spiroplasma showed a close relationship with crab spiroplasma with 99% identity (Table 2) it is premature to conclude that both isolates are the same species, since previous experimental infections had failed to transmit TDA to the crayfish . Further studies are being performed to determine the exact taxonomic positions of the two microbes.
Spiroplasmas are helical, wall-less procaryotes classified within the family Spiroplasmataceae, of the order Entomoplasmatales, class Mollicutes, phylum Firmicutes in the domain Bacteria [12–14]. They are unique in their ability to maintain their helical shape without a cell wall. The most recent revision describes 34 spiroplasma groups and 14 subgroups . Since spiroplasmas were first detected in a plant disease (stubborn disease), more and more spiroplasmas have been found in plants and insects over a wide global geographic range [16–19]. Some plant spiroplasmas, including Mulberry crinkle spiroplasma, barley stunt spiroplasma and Hibiscus syriacus spiroplasma and insect (bees) spiroplasma have been isolated and confirmed in China [20–24]. Spiroplasmas in freshwater animals had not been found any where until the discovery of the spiroplasma in Chinese mitten crab . The confirmation of spiroplasmas in another freshwater animal and crustacean, red swamp crayfish, provokes the attention that spiroplasmas exist not only in terrene but also in freshwater environments and may have the diffusibility among the freshwater crustacean. Further studies should be performed to ascertain the potential menace to other species in freshwater animals.
An important consideration from the studies is that both PCR and TEM methods showed positive results in some bottom mud of the ponds associated with the disease, whereas water samples were negative. All freshwater crustaceans, including crayfish and crab, commonly dwell in the bottom, therefore the bottom mud could be the important reservoir for spiroplasma living outside their hosts. Although some results from PCR from bottom mud coincided to that of TEM observations with negative staining, there were still some conflicting results with some positions showing positive in PCR but negative in isolation in M1D. The explanation could be the agents in some positions were too few to be cultivated. Compared to the method of isolation in M1D medium, the PCR method is more sensitive and effective for detecting spiroplasma and can be used for environmental detection of spiroplasma in freshwater ecology.
Besides freshwater crustacean, two marine shrimps, Alvinocarid shrimp (Rimicaris exoculata) and Penaeus vannamei, have been found associated with spiroplasmas. The spiroplasma in Alvinocarid shrimp appears to be part of the normal gut flora , while the spiroplasma in Penaeus vannamei is pathogenic and the causative organism of the epizootic in Colombia . It is obvious that spiroplasmas have harmful impact on water animals both in freshwater and marine environments. These findings are not only significant for studies on pathogenic spiroplasmas, but also have implications for studies of freshwater and marine ecology.
Red swamp crayfish, Procambarus clarkii, have been studied mainly for burrowing activity [27,28] and metals accumulation (Generally, crustaceans accumulate some metals directly proportional to the increase in bioavailability from water and food-chains) . This paper provides a study on a novel agent, spiroplasma, which may present a great potential as a disease agent in the freshwater environment. Based on our present work and earlier studies on the TD of Chinese mitten crab we conclude that spiroplasmas play an important role in the epizootics in freshwaters species.
Thanks are due to Mr. Xu Zaikuan for helping sample crayfish in commercial growout ponds, Mr. Du Kaihe for providing technical assistance for electron microscopy and Mr. Long Yi for PCR technical assistance. We also thank Dr. Linda Nunan for correcting the manuscript. This work was supported by grants from the NSFC (No. 30371118) and the Natural Sciences Research Fund of Sciences and Techniques Office in Jiangsu Province (No. BK2002024).