in Revista MVZ Córdoba
Detection of Perkinsus sp. in Chionista fluctifraga cultivated in the southeast Gulf of California
Abstract
Objective. To determine the presence of Perkinsus sp. in the sand clam Chionista fluctifraga cultivated in an intertidal zone of the southeastern Gulf of California, using Ray’s thioglycollate fluid medium staining (MFTR) and polymerase chain reaction (PCR). Material and Methods. The prevalence and parasite load of Perkinsus sp. were monthly obtained and correlated with environmental water conditions and clam biometric indicators (n=540), from May 2018 to September 2019. Results. Presumptive hypnospores of the protozoan were detected in eight months of culture. The prevalence fluctuated from 3.3% to 13.3%, while the average parasite load ranged from 2 to 1286 hypnospores/g of tissue. The intensity of infection varied from negative to light. The prevalence and parasite load were correlated with each other (r=0.61, p<0.05) but not with the environmental parameters, nor with the biometric indicators of the clam. The PCR test was negative for the MFTR positive cases. Conclusions. Presumptive hypnospores of Perkinsus sp. were detected in C. fluctifraga without clear indications of infection that compromised the clam health in culture. The constant monitoring of Perkinsus sp. in C. fluctifraga is highly recommended to establish possible infections.
Main Text
INTRODUCTION
The mollusk industry represents an important source of food worldwide. According to the Food and Agriculture Organization of the United Nations (FAO), its cultivation contributes approximately 3.5 per cent (17.1 million tons) of its total annual production (1). In Mexico, several clam species are valued both for their demand for human consumption and for their aquaculture potential (2,3). However, one of the main “bottlenecks” in the development of their crop is disease.
Perkinsosis is a disease that has affected the world’s bivalve mollusk industry (4), which is caused by different species of the Perkinsus protozoan and has sometimes been associated with massive mortalities (5). Such is the negative effect it causes on the cultivation of mollusks that this pathogen is considered by the World Organization for Animal Health as a parasite subject to compulsory declaration (6). In Mexico, Perkinsus marinus was reported infecting the Eastern oyster Crassostrea virginica in the Gulf of Mexico (7) and, for about two decades, the Cortez oyster Crassostrea corteziensis in the Mexican Pacific (8). For a few years, protozoa of the genus Perkinsus have been detected in various species of wild bivalve populations within the Gulf of California, such as the atrina maura(9) and the chocolate clam Megapitaria squalide (10). In cultures, the presence of P. marinus was confirmed in the smooth clam Chionista fluctifraga along with the Japanese oyster Crassostrea gigas on a farm in the northeast Gulf of California (11), confirming the horizontal dispersal of the pathogen between species in the area.
Recently, C. fluctifraga was polycultured with C. gigas and M. squalida, in an intertidal zone on the southeastern coast of the Gulf of California, with promising harvest time results (12). However, the health status of its culture in relation to the presence of this parasite is unknown, which would pose a risk to its production and other species (3). The purpose of this study is to evaluate the presence of Perkinsus sp. in smooth clam grown in El Colorado Bay, Ahome, Sinaloa, by detection and confirmation with the standard Ray Thioglycolate Fluid Medium (MFTR) technique and polymerase chain reaction (PCR). the indicators (prevalence, parasitic load, and intensity of infection) and their relationship with environmental conditions and clam biometrics are determined respectively.
MATERIALS AND METHODS
Collecting organisms and recording water parameters. Monthly (April 2018-September 2019), 30 clams of Chionista fluctifraga were collected from the intertidal culture plot belonging to the company SEA FARMERS S.A de C.V, located in El Colorado Bay, municipality of Ahome, Sinaloa. The plot is delimited by the following coordinates: 25° 43’ 46.30”-25° 43’ 38.70” N and 109° 33’ 54.50”-109° 23’ 47.80” W.
The specimens were transported alive in a container with sea water and aeration for one hour, to the Malacology Laboratory of Instituto Politecnico Nacional - Centro Interdisciplinario de Investigacion para el Desarrollo, Integral Regional (IPN-CIIDIR), under the standard rules (NOM-031-SSA1-1993, Goods and Services. Fishery products. Fresh- cooled and frozen bivalve mollusks). It should be noted that the Ethics Committee of the IPN-CIIDIR Teacher’s Association authorized this research.
Also, in each sample, the temperature and dissolved oxygen of the water were obtained with an oximeter (YSI,5/12FT), pH with a potentiometer (HANNA), salinity with a precision refractometer (ATAGO), and depth and transparency with a Secchi disk. Total suspended solids (SST), particulate organic matter (POM) and chlorophyll A concentration (Cl-.) were determined according to the gravimetric (13) and spectrophotometric (14) method for sea water.
Biometric and infectious indicators. For each clam, the length (maximum distance between the anterior and posterior margins), height (maximum distance from the umbo to the ventral margin), and the width of the shell (maximum distance between the thickest parts of the two shells) were obtained with a digital vernier ruler (0.01 mm), plus total weight and soft tissue weight (0.01 g, wet weight) with a portable balance. Soft tissue was processed according to the standard Ray Thioglycolate Fluid Medium (MFTR) technique to detect suspected Perkinsussp. hypnospores. (6). Prevalence (number of clams with suspected hypnospore/total number of clams) X 100 (15), parasitic load = number of hypnospore per gram of clam tissue (16), and intensity of infection categorized as: Negative (0 hypnospores), mild (<104 hypnospores per gram of tissue), moderate (104 to 105 hypnospores per gram of tissue), and severe (>105 hypnospores per gram of tissue) (17).
For digestion, 4 g of clam tissue in tubes with MFTR was used to be incubated in darkness for 7 days at room temperature. When the tissue weighed less than 4 g (small clams), the whole body was used in digestion. Subsequently, 35 mL NaOH 2M was added to digest for 60 min at 60°C. Finally, the tubes were centrifuged (2.000 rpm per 3 min), NaOH 2M was discarded and rinsed with distilled water conserving the sediment of the sample (6). For DNA extraction from spores, samples are washed with distilled water and, under a microscope (10X), the spore wall is broken with a fine-pointed needle. Finally, 1 mL of DNAzol® and 5 µL of proteinase K (6) are added to the sample (approximately 100 µL) .
DNA obtained from digestions and extractions of both, hypnospores and gill tissue, was used as white DNA in the electrophoretic PCR reaction (6). Oligonucleotides PerkITS85 (5’-CCG-CTT-TGT-TTG-GAT- CCC-3’) and PerkITS750 (5’-ACA-TCA-GGC-CTT-CTA-ATG-3’) were used to detect any species of Perkinsussp. (18), and the specific oligonucleotides PmarITS-70F (5’-CTT-TTG-YTW-GAG-WGT-TGC-GAG- ATG-3’) and PmarITS600R (5’-CGA-GTT-TGC-GAG-TAC-CTC-KAG-3’) for P. marinus (19).
Genomic DNA from C. corteziensis infected with P. marinus (20) was used as a positive control, and water was added instead of DNA as a negative control.
Statistical analysis. Data that did not show a normal distribution (environmental parameters and infectious indicators, Kolmogorov- Smirnov) were transformed (square root) before statistically analyzing them. Multivariate correlations (., Pearson) were applied between the infectious indicators and the rest of the parameters (environmental sampling site and biometrics corresponding to the clam) with a significance level of 0.05. The Statgraphics Centurion XVII program was used.
RESULTS
The ranges of water parameters were: Temperature = 15.9°C in February 2019 to 32.1°C in September 2019; salinity = 25 g salt/L in September 2018 to 40 g salt/L in March 2019; dissolved oxygen = 5.14 mg/L in August 2018 to 9.63 mg/L in April 2018; PH = 4.3 in August 2018 to 8.21 in March 2019; depth = 0.20 m in March 2019 to 1.15 m in September 2019; transparency = 0.16 m in April 2019 to 0.8 m in October 2018 and September 2019; CL-. = 2.2 mg/m3 in August 2019 to 10.5 mg/m3 in November 2018; SST = 19.3 mg/L in July 2018 to 189.2 mg/L in November 2018; and MOP = 4.8 mg/L in June 2018 to 26.3 mg/L June 2019. Table 1 shows biometric values of the clam for each month.
Prevalence and parasitic load had an interval of 0% to 13.33% and 2 to 1.286 hypnospores per gram of tissue (Figure 2). The intensity of infection in all months of cultivation was zero (April, July, September, October, November and December 2018, and January, February, March, May, June, July, august, and September 2019) to light (may and August 2018, and April 2019).
Of the 19 clams that were positive for MFTR staining (3.5%), no confirmation was obtained for Perkinsus sp. or P. marinus with the PCR test. There was no correlation between the infectious indicators with the water and biometrics parameters of the clam, but between the prevalence and parasitic load (.= 0.61, p<0.05).
DISCUSSION
Although the recoded environmental parameters were whithin the range for clam cultivation (21), our values recorded a wider range to work reported in the same bay (22,23). This can be explained, in part, to the duration of the experimental times between investigations and to the inherent conditions of an intertidal zone culture, which suffers cyclical exposure to the weather. On the other hand, biometrics showed a constant growth of C. fluctifraga during the 18 months (12), with no signs of involvement due to the environmental indicators recorded.
The different soft tissue components of C. fluctifraga showed no apparent characteristic Perkinsosis damage (watery and thin tissue, slow closing of leaflets and whitish corpuscles, 6) under visual and microscope observation. However, MFTR staining detected suspected protozoan hypnospores in clam tissue 8 months after culture, without a trend dictated by environmental parameters. This is consistent with the reports of other authors (20,27) studying various bivalves near the study area, who mention that infectious indicators were not correlated with fluctuations in water parameters, including temperature and salinity, the main indicators recognized as promoters of infection (4). The detection of hypnospores of Perkinsussp. and Perkinsus marinus has previously been reported in different bivalve species in the Gulf of California in Mexico (9,10), including the smooth clam (11), but its presence has not been associated with serious infections or mortalities. The average prevalence of Perkinsus sp. in C. fluctifraga obtained with MFTR (3.5%) was lower than that reported in a crop located north of the east coast of the Gulf of California for the same species (11), As for other bivalve mollusk species in the Gulf of California (8,24). In addition, the average parasitic load of Perkinsus sp. in 18 months of smooth clam culture (195 hypnospores/g), associated with a slight intensity of infection, suggests that: 1) the environmental conditions of the site have favorable health characteristics for its cultivation (25), which is consistent with its classification as a certified area for the cultivation of mollusks, and, 2) in agreement with other clams (10,26) studied near the study area, C. fluctifraga shows no tissue damage from this pathogen (4).
The prevalence and parasitic load of the protozoan were not correlated with any environmental or biometric host parameter, which is consistent with that reported for C. gigas grown in the same bay (27). It is therefore possible to infer that the conditions of the site have remained stable, without affecting the health condition of the bivalves studied in that site. However, the results of this study are different from those reported for wild species of M. squalida south of the culture site, where the protozoan infectious indicators did show correlation with the size of the chocolate clam and salinity (10,26). The difference in results with wild adult organisms could be explained by the longer period of time they have been exposed to the parasite before being sampled, in addition to the environmental conditions of each zone.
Despite the sensitivity and specificity of the PCR technique (6), the pathogen was not detected, probably due to the low values of the infectious indicators expressed in little or no genetic material (DNA) available for testing. this coincides with the degree of infection intensity of the clams, which was from zero to light. Staining with thioglycolate showed suspected parasite hypnospores, but with low prevalence and number of suspected hypnospores per gram of tissue. The difference found in both MFTR and PCR can be attributed to the amount of tissue tested in each test. For the thioglycolate staining test, 4 g of tissue was used, whereas for the PCR test, approximately 50-100 mg was used in DNA extraction. The same was observed for other bivalve species in the region (9,27,28).
Since C. fluctifraga grew steadily, with no signs of infection, it is possible to conclude that the health status of its crop in the intertidal zone of the southeastern Gulf of California would not be compromised by the presence of Perkinsus sp. However, due to its dispersal and detection in different bivalves of the area, constant monitoring of the parasite in future crops and their surrounding areas is recommended.
Conflict of interest
The authors declare that there was no conflict of interests of any kind during the realization and elaboration of this work.
Abstract
Main Text
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION