Study area
Galerazamba (10° 47' 38'' N, 75° 14' 48'' W): is a 220 ha thalassohaline saltwork with five ponds, three for brine and two for crystallizers. It is located approximately 20 km North of Cartagena city, at the borderline of the Bolívar department (Fig. 1). Studies have been conducted by several authors in the past using samples from this location[5, 11–15, 32, 35, 43–55]. This saltwork, built in a natural saline lagoon and surrounded by mangroves, formed by a sandy-clay and loamy-clay soil type, floods with seawater during high tide throughout the year [45–47].
Salina Cero or Ciénaga Prieto (10° 46' 29''N, 75° 15' 55''W): is an 18 ha thalassohaline lagoon 3 km of Galerazamba, Bolivar department (Fig. 1)[11, 12, 35], studied in September 1998. For many decades, salt has been manually extracted once or twice per year, and fishermen noted the presence of Artemia for over five decades.
Kangarú (11° 59' 28''N, 74° 32' 21''W): is less than a 4 ha natural thalassohaline saltwork comprised of three small ponds located in the northern region of the Salamanca Island National Natural Park, Magdalena department (Fig. 1). It was explored in July 2000. Salt has been occasionally exploited for decades. This locality, is an important bird migration spot, however, it lost importance because of mangrove destruction as consequence of building a highway through the park.
Pozos Colorados (11° 09' 45''N, 74° 13' 34''W): is an approximately 65 ha very old artificial thalassohaline saltwork, currently abandoned. Few studies have been conducted by local researchers in the past using samples from this location. It is located near Santa Marta city, Magdalena department (Fig. 1), contiguous to the road connecting to Barranquilla to Santa Marta city [11]. This saltwork consists of only five irregularly shaped, shallow ponds with only 4 ha water surface.
Tayrona National Natural Park (Chengue natural saltwork where the 'Tayrona' Artemia population was first reported) (11° 19' 03''N, 74° 08' 13''W): This natural thalassohaline saltwork (Fig. 1) of approximately 2.5 ha is hypersaline due to a closure pattern of dynamic sedimentation of the communication channel with the inlet [54]. It is located in the Magdalena department [11, 12, 35, 53–56]. Tayrona NNP encompasses a small number of saline non crystallizing ponds, with the exception of Chengue, where Artemia has been reported to occur. The salt pond is flooded during most of the year and serves as a saltwork during summer [57]. Chengue Inlet, is located in the middle of the Tayrona NNP, it presents a series of small bays and inlets extending from Santa Marta to Cañaverales to the east. Chengue salt exploitation existed long before the prehispanic period [58].
Manaure (11° 46' 32''N, 72° 29' 27''W): is located to the west, contiguous to the town of Manaure, in the center of La Guajira department, near Riohacha city (Fig. 1). Studies have been conducted by several authors in the past using samples from this location [5, 11–13, 35, 43, 47, 48, 50, 52, 54, 55, 59]. This saltwork is a thalassohaline, shallow water body extending over 4,000 ha. Water movement through the saltwork system is achieved both by pumping and through gravity. There are six pumping stations that increase water volume to a predetermined water level, thereafter water will flow by gravity. This zone was originally a natural lagoon surrounded in some areas by mangroves. The deposits were constructed using the natural topography of the terrain with some modifications. The levees were built by compacting large amounts of clay material brought from the margins of the saltwork [47].
Warrego (12° 19'N, 71° 54'W): is an approximately 600 ha (2 miles long) thalassohaline saltern located in the northern tip of La Guajira department, near Puerto Nuevo village (Fig. 1). Occasionally, the Wayu Indians extract salt when the brine crystallizes. Since it was completely dried up when we visited it (January 18, 2000), no water samples were collected from this location and found few Artemia cysts.
Bahía Hondita (12° 19' 28''N, 71° 44' 13''W): is a natural thalassohaline saltern, approximately 3000 ha, located in La Guajira department (Fig. 1). The Wayu Indians also extract salt in this saltern when the brine crystallizes. We visited the area on January 18, 2000 and only found Artemia cysts.
Pusheo (12° 20' 47''N, 71° 44' 17''W): is an approximately 400 ha thalassohaline saltern located in the northern tip of La Guajira department (Fig. 1), near Punta Gallinas. Occasionally, the Wayu Indians extract salt when the brine crystallizes. We visited the area on January 18, 2000, and only found Artemia cysts.
Preparation and sampling
Sampling was conducted monthly and cysts batches were collected irregularly (whenever available) in nine thalassohaline locations aforementioned in the northern region of the Colombian Caribbean, from July 1998 to June 2000. Cyst processing was done following these steps: (i) size separation with brine, (ii) density separation in brine, (iii) washing in freshwater, (iv) density separation in freshwater, (v) drying below 40°C, and (vi) vaccum packing and refrigerating cyst at 4 ± 2°C.
Cyst diameter and chorion thickness were recorded from sites where sufficient cysts were collected, using SFB (USA, ARC1258) cysts as reference material. Cysts were incubated for 3 hr in 10 g.l-1 artificial sea water (Instant Ocean®) at 25 ± 0.5°C and pH 8.3 [13]. One percent lugol's solution (5 %) was added to the sea water to stop embryos from hatching and cysts were in the dark overnight. Cyst diameter (μm) was measured in 200 cysts with a precalibrated microscope. Mean value and standard deviation were calculated using the predetermined conversion factor. Decapsulated cyst diameter (μm): a small sample of cysts was hydrated in tapwater for 2 h. Cysts were then decapsulated with a NaOH and NaOCl solution. Cysts were rinsed well and incubated in 10 g.l-1 artificial sea water (Instant Ocean ®) with 1 % lugol for 1 hr, at 25 ± 0.5°C, and pH 8.3 and was incubated for 1 h more. Afterwards, 1 % lugol was added again to the incubating solution and cysts were left overnight in the dark.
Decapsulated cyst diameter was measured for 200 cysts with a precalibrated microscope. Mean value and standard deviation were calculated using the predetermined conversion factor. Chorion thickness was calculated using this formula:
(cyst diameter - decapsulated cyst diameter)/ 2
Naupliar length was determined on Instar I nauplii, following this procedure [13]: cysts were incubated and hatched under controlled conditions (25 ± 0.5°C, pH 8.3 and illumination: 1000 lux) in artificial sea water (Instant Ocean®) at 35 g.l-1 salinity [32]. Nauplii were sampled at Instar I considering the protopodite of each antennae which bears two endites with a single long bristle attached to each and their brownish-orange color due to yolk presence (Instar II is translucent) as the traits defining this stage [60, 61]. Nauplii were harvested when 90% of the total number of hatchable nauplii had been produced [22]. Two hundred nauplii were fixed in lugol's solution (5%) and the length determined using a microscope with a pre-calibrated projection system. Cyst quality studies [13] were performed only on major saltworks. The following parameters were used to evaluate cyst quality:
i) Hatching percentage (H%): number of nauplii that can be produced under standard hatching conditions from 100 full cysts (with embryos).
H% = (N × 100)(N+U+E)-1
Cysts (1.6 g) were incubated in 800 ml of 32 g.l-1 microfiltered (<1 μm) seawater (Instant Ocean®) under continuous illumination (2000 lux) at 28°C, pH = 8.3, in a cylindroconical vessel (test was run in triplicate per strain) with bottom aeration (>2 mg.l-1). Vessels were suspended in a water bath in a 100 gal aquarium with a water heather and a mixer to maintain a well distributed temperature (± 1°C). After 24 h incubation six 250 μl subsamples were taken from each cone with a micropipet. Each subsample was pipetted into a small vial and nauplii were fixated by adding a few drops of lugol's solution (5%). Nauplii (ni) and umbrella (ui) stages were counted in each subsample under a disection microscope. Mean values (N = nauplii and U = umbrella) were calculated each for these two stages. Unhatched cysts were decapsulated and empty cyst shells were dissolved with a drop of NaOH solution (40 g.100 ml-1 distilled water) and five drops NaOCl (5.25% NaOCl) added to each vial. Unhatched (orange colored) embryos (ei) were counted per cone (i = 6) and mean value (E) was calculated for each cone. H% value was calculated per cone, and mean value and standard deviation was calculated for three cones (final H% value).
ii) Hatching efficiency (HE): number of nauplii/g dry cysts that can be produced under standard hatching conditions.
HE = (N × 4 × 800 ml)(1.6 g)-1
HE value was calculated, for each strain evaluated, per cone, and mean value and standard deviation was calculated for three cones (final HE value). Hatching vessels were left for another 24 h, subsequently subsamples were again taken to calculate H% and HE for 48 h incubation period.
iii) Hatching rate (HR): period from incubation (cyst hydration) to nauplii release (hatching). The following HR time intervals are considered:
T0 = Incubation time untill appearance of first free swimming nauplii
T10 = Incubation time untill appearance of 10 % of total hatchable nauplii
T90 = Incubation time untill appearance of 90 % of total hatchable nauplii
TS = T90 - T10 ; this value gives an indication of the hatching synchrony
Six 250 μl samples, for each strain evaluated, were taken 12 h after incubation and HE was calculated every 3 h until HE mean value remained constant for three consecutive sampling periods. Mean values per period were then expressed as percentages of the maximal HE. A hatching curve was plotted for each strain, and T10 and T90 were extrapolated from the graph.
iv) Number of cysts.g-1: this parameter is dependent on cysts diameter. Cysts (4 g) were placed in an aluminum plate and weighted and dried in a drying oven set at 60°C for 24 h. Cysts were then cooled down to room temperature for 4 h in a tightly sealed glass drying chamber with fresh desiccant. One g of cyst sample (triplicates) was then weighted (0.1 mg accuracy) in an aluminum plate; to determine average cyst weight, for a single cyst, ten subsamples (10.0 ± 0.1 mg) were taken from each replicate and counted; to find how many cysts were in the 1 g sample average cyst number in 10 mg was then extrapolated to the 1 g cyst sample. This procedure was repeated for each strain evaluated.
At each location we measured: salinity (temperature compensated refractometer), percent O2 saturation and temperature (Oxymeter WTW® 330), pH (pH meter WTW® 330), nitrates, nitrites and phosphates (Hatch® DREL 2010 spectrophotometer), and chlorophyll a. For the latter, we used the Seston method and read (Hatch® DREL 2010 spectrophotometer). The ionic composition (Table 3) was determined using a Unicam 939/959 atomic absorption spectrophotometer. All samples were diluted with deionized water because of the high ionic concentration. A sample of nauplii from Galerazamba, Manaure, Salina Cero and Tayrona was taken for FAME; for this analysis we followed Sorgeloos et al. [13]: cysts were incubated and hatched under controlled conditions (25 ± 0.5°C, pH 8.3 and illumination: 1000 lux) in artificial sea water (Instant Ocean®) at 32 g.l-1 salinity. FAME methodology for freshly hatched nauplii (0.25 g) was a modification of the direct esterification described by Lepage and Roy [62]. The latter implicates a direct acid catalized transesterification without prior extraction of total fat, on dry sample (triplicates) amounts ranging from 10 to 150 mg. An internal standard 20:2 (n-6) was added prior to the reaction. FAME were extracted with hexane. After solvent evaporation FAME were prepared for injection by redissolving them in iso-octane (2 mg/ml). Quantitative determination was done by a Chrompack CP9001 gas chromatograph equipped with an autosampler and a TPOCI (Temperature programmable on-column injector). Injections (0.5 μl) were performed on column into a polar 50 m capillary column, BPX70 (SGE Australia), with a diameter of 0.32 mm and a layer thickness of 0.25 μm, connected to a 2.5 m methyl deactivated precolumn. The carrier gas was H2, at a pressure of 100 kPa and the detection mode FID. The oven was programmed to rise from the initial temperature of 85 to 150°C at a rate of 30°C/min, from 150 to 152°C at 0.1°C/min, from 152 to 172°C at 0.65°C/min, from 172 to 187°C at 25°C/min and to stay at 187°C for 7 min. The injector was heated from 85 to 190°C at 5°C/sec and stayed at 190°C for 30 min. Identification was based on standard reference mixtures (Nu-Chek-Prep, Inc., USA). Integration and calculations were done on computer with a software program Maestro (Chrompack).
Any experimental research on animals that was reported in this study was performed with the approval of an appropriate ethics committee regulating animal research.
Calculations and statistics
Standard deviations were calculated for all cyst diameter and naupliar length measurements. Data obtained were analyzed using one-way ANOVA, and averages compared with Duncan's test (SPSS V10.0).