Water samples were collected at two sites (reservoir and pond) within the Finca Doña Luisa S. de R.L. de C.V. shrimp farm and two sites (northern mouth and El Tortugón estuary) in the Macapule lagoon. The lagoon sampling stations were separated ~9 km from each other and 2.1 km and 10.8 km from the shrimp farm, respectively (Fig. 1).

Figure 1 Source: The authors.

Water samples between 4 and 10 L at the lagoon were collected monthly from December 2007 to December 2008 using a segmented tube; ~1 L samples at the farming facility were collected manually from the surface (0.5 m depth) in June 2007, and monthly from March to November 2008.

Samples were sieved through a 200 μm-mesh net and then separated into size fractions using a series of graded filters: Nuclepore polycarbonate membranes (20 and 0.2 μm), GF/C glass microfiber filters (1.2 μm), and Anodisc aluminum oxide membrane filter (0.02 μm). The volume of seawater filtered ranged from 20 mL for the smaller pore sizes to 1000 mL for the larger ones.

DNA was extracted from seston retained in the filters using DNAzol™ reagent (Invitrogen™), following the manufacturer’s instructions; a total of 107 DNA extractions were obtained.

To assess the quality of DNA extracts, we amplified 16S rDNA with the universal primers F2C (5′-AGAGTTTGATCATGGCTC-3′) and C (5′-ACGGGCGGTGTGTAC-3′) in those availed extracts from both lagoon and pond, collected from December 2007 to July 2008 [¹⁶]. PCR amplification thermal conditions consisted of 94 °C for 4 min (initial denaturation) and 32 cycles at 94 °C for 30 s (denaturation), 60 °C for 30 s (annealing), 72 °C for 2 min (extension), and 72 °C for 5 min (final extension). The WSSV was initially amplified using a standard nested PCR assay [¹⁷], following the thermal conditions at 94 °C for 4 min (initial denaturation), 94 °C for 30 s (denaturation), 55 °C for 30 s (annealing), 72 °C for 1 min (extension), and 72 °C for 5 min (final extension), with 30 and 35 cycles for the one-step- and nested-PCR assays, respectively, from denaturation to extension step. These amplifications were confirmed with two commercial kits, namely IQ₂₀₀₀ ^TMWSSV Detection and Prevention System (based on nested PCR assay) and IQ_REAL ^TMWSSV Quantitative System (based on real-time PCR assay) following the manufacturer’s instructions (GeneReach Biotechnology Corp.).

All amplified products from IQ₂₀₀₀ ^TMWSSV Detection and Prevention System were mixed with a loading buffer [0.25 % (w/v) bromophenol blue, 0.25 % (w/v) xylene cyanol F.F., 30 % glycerol in distilled water], separated by electrophoresis on 1 % agarose gel stained with ethidium bromide (0.5 mg/mL) in 0.5X T.B.E. buffer at 70 V, and then visualized using a U.V. trans-illuminator. A 1 kb molecular-weight size marker was loaded into the gel and the PCR products to identify the approximate size of the amplified fragments. Based on the viral load found in positive samples amplified with the IQ₂₀₀₀ ^TMWSSV protocol, three possible migration patterns were expected on the agarose gel: three (910, 550, and 296 bp), two (550 and 296 bp), or a single (296 bp) band, which would indicate a high (>2000 copies/reaction), moderate (>200-2000 copies/reaction), or low (>20-200 copies/reaction) WSSV concentration, respectively. WSSV copies in the TaqMan IQ_REAL ^TMWSSV assay products were quantified (absolute values) with the equation generated from the threshold cycle that relates Cq values to the log template amount. The viral concentration in the WSSV-positive extracts, as viral copies per volume (mL), was estimated by considering the filtered volume of seawater for DNA extraction.

Only two (1.2 and 20 μm fractions collected in the shrimp pond in June 2007 and July 2008) of the 107 DNA extracts tested with the standard nested PCR assay proposed by Kimura et al. (1996) were positive for WSSV (Fig. 2). Both nested PCR results were confirmed by IQ₂₀₀₀ ^TMWSSV diagnostic kits, but the IQ_REAL ^TM assay returned a WSSV negative result for the June 2007 sample. When considering the filtered water sample volume for DNA extraction in the results of this quantitative assay, a viral concentration of 6.54 × 10⁴ WSSV copies/mL was estimated for the July 2008 sample, which is 2 to 3 orders of magnitude larger than the semiquantitative kit IQ₂₀₀₀ ^TMWSSV (Fig. 3).

Figure 2 Source: The authors.

Figure 3 Source: The authors.

This study applied methodological procedures commonly used to diagnose WSSV in animal tissues to seston samples from a shrimp farm and natural environments. Sample storing and handling (e.g., during the removal of potential inhibitors from seawater) may have affected DNA extraction, leading to our inability to detect the virus in most samples; thus, caution is advised in interpreting whether a sample was WSSV positive or not.

High concentrations of phenolic compounds, heavy metals, humic acid, or urea are the most common agents in environmental samples interfering with PCR detection (e.g., [^18-20]. Of these compounds, only urea has been found in high and variable concentrations in the water column at Macapule Lagoon [²¹], although the significant levels of heavy metals in soft tissues of bivalves might be a potential pathway from sediments and settling seston [²²].

Assuming that prokaryote DNA is ubiquitous and more abundant than WSSV DNA in seston size fractions, the results of the DNA quality test in extracts from both Lagoon and pond samples (n = 92) showed that only 50% of them produced clear single bands on the agarose gel electrophoresis. Thus, DNA preservation and inhibitory components in these samples were not likely to affect the WSSV test results obtained with nested PCR. The relatively low volume of filtered seawater may have been another contributing factor in the recurrent negative detection of WSSV. However, water samples (~20 L, collected at the estuary in March 2009) that had been pre-concentrated by tangential filtration (>0.02 μm) tested negative for WSSV with nested PCR.

The WSSV positive results showed that the 1.2 and 20 μm fractions could be a potential route for transmitting this virus to crustaceans by either direct contact or ingestion by filter feeders. Inert and biological particles in this size range present in coastal lagoons mainly include resuspended sediments, self-assembled organic colloids (microgels), and nanoplankton. It is known that WSSV can remain viable in shrimp pond sediments for up to 19 days under sun-drying conditions and up to 35 days in undrained sediments [^23,24].

Clay and very fine silt are the dominant sediment fractions in the Macapule Lagoon [²⁵], and their resuspension into the uppermost water layer is facilitated by wind-induced turbulence, currents, and tides flowing over the topographic features [^21,26,27]. Consequently, WSSV attached to sediments may play a significant role in the outbreak dynamics when they become resuspended or, indirectly when they come into contact with eggs of planktonic organisms and invertebrates that shrimp consume [²³].

Self-assembled microgels are abundant in productive systems and act as microbial activity hotspots and reservoirs of detritus, small cells, and viable viral particles [²⁸]. Highly “sticky” fractions of such microgels, which were retained by 0.4 μm pore polycarbonate membranes and quantified with the Alcian Blue staining method, were found in high concentrations (range 0.11 to 49 μg GX eq. ml^-1; for methods see [²⁹] in seawater samples from Ohuira coastal lagoon, near our study site. However, no experimental data are currently available to support the potential association between self-assembled microgels and the viability and transport of WSSV.

A wide range of planktonic organisms (e.g., microcrustaceans, rotifers, and microalgae), benthonic crustaceans, and Polychaeta worms that are not WSSV infected have been reported as carriers [^30,31]. Some life stages of these organisms may be present in both plankton and benthos, increasing the probability of acquiring and spreading the disease. In shrimp farms and other Mexican coastal systems, including the Macapule lagoon, the most frequently observed potential vectors of WSSV in meso- and macro-zooplankton belong to non-crustacean groups, such as chaetognaths and fish larvae [^32,33]. Esparza-Leal et al. [⁶] monitored 12 ponds in a shrimp farm at Guasave City for two weeks after a WSSV outbreak and found WSSV-positive results in the >10 μm and >0.45 μm plankton fractions from one (#7) and three (#2, #7 and #10) ponds, respectively. The same study conducted infectivity assays on healthy shrimp differentially exposed to ten-sized fractions (0.1 to 100 μm) of filtered water and particulate material from a pond after a WSSV outbreak. They found positive detection after 216 h in shrimp that had been exposed to the liquid fractions: <40 μm, <10 μm, and <0.65 μm; and the particulate fractions: >100 μm, >40 μm, and >5 μm. Moreover, not all replicates from the same fractions tested positive for WSSV. This variability was attributed to the relatively high temperature (30‒33°C) in the experimental systems, which might have inhibited WSSV replication [³⁴].

Nanoplankton (encompassing phyto- and zooplankton) is one of the most prolific and essential components in the planktonic food web structure in natural and artificial systems influenced by enrichment conditions. The average nanophytoplankton abundance in the Macapule lagoon and the shrimp culture system during the study period was 7.90 ± 5.52 × 10³ and 2.13 ± 1.14 × 10⁴ cells/mL, respectively [^21,35]. The environmental conditions in ponds favor autotrophic production and microbial abundance, thus increasing the probability of encounters between nanoplankton cells and hosts releasing WSSV particles. This association partially supports the empirical model of viral transmission to higher trophic levels by virus attached to phytoplankton [^8,36], although this hypothesis has not been tested in samples from natural environments.

Moreover, the possibility that coastal ecosystems function as temporary or permanent WSSV reservoirs due to the discharges of untreated pond wastewater cannot be ruled out, as reported by several investigations elsewhere [^9,10,37,38]. Discharge operations are carried out during the shrimp harvesting season with no wastewater sanitization/treatment. Besides potentially affecting the local diversity of crustaceans, the virus could likely be reintroduced and infect animals in the aquaculture system during seawater supply. Moreover, post-tropical storm conditions - i.e., high temperature and high ammonia concentration, low density of heterotrophic bacteria, and low oxygen concentration - have been identified as triggers of WSSV outbreaks [^5,39]. Thus, the weather might play a key role by modifying currents, precipitation, and temperature conditions, among other ambient conditions.