DEVELOPMENT OF A SIMPLE METHOD TO INOCULATE NECROTIZING HEPATOPANCREATITIS BACTERIUM IN ARTEMIA SP

Different protocols were performed to evaluate their effectiveness to inoculate necrotizing hepatopancreatitis bacterium (NHPB) into Artemia sp. Protocol-A consisted of adding to water 0.2 mL·L-1 of bacterial inoculum composed of infected hepatopancreas (HPNHPB) and glycerol (1:1 w/v). Protocol-B consisted of adding the same inoculum but at a ratio of 2 mL·L-1, whereas protocol C consisted of adding 0.2 mL·L-1, of HPNHPB+glycerol+formulated feed (0.5:0.5:1 w/v/w). Controls for each treatment consisted of similar inoculums but using healthy hepatopancreas. Artemia from A and B treatments were negative for NHPB after four and eight days post-inoculation, whereas Artemia from treatment C resulted as positive to NHPB after four (75% of individuals) and eight (100%) days post-inoculation. The results suggest that the incorporation of formulated feed into the inoculums induced the bacteria’s consumption of Artemia.


INTRODUCION
Branchiopod crustaceans have been reported as disease transmitters within aquatic ecosystems (Holdich and Pöckl 2007).In particular, Artemia sp. is widely used in the aquaculture of fish and crustaceans; additionally, they are commonly observed in diverse aquatic habitats, being a food source for diverse aquatic animals (Campaña-Torres et al., 2010).Thus, it is possible to hypothesize that Artemia could play a role as a transmitter of pathogenic organisms; in fact, some authors have demonstrated that Artemia is a vector for some virus and bacteria (Cano et al., 2009;Lone et al., 2009).
Several laboratory studies have been conducted to evaluate zooplankton species as possible disease vectors.However, it is difficult to perform certain laboratory experiments in order to know the role of Artemia or other small size organisms in the spread of pathogenic virus and bacteria because they cannot be inoculated by forced feeding or injection as can be done with larger animals.Thus, the few strategies to achieve such a goal include promoting the voluntary consumption of inoculums by Artemia and/or, inducing a physical contact among the pathogen organisms and Artemia.
Developing a simple but effective protocol to inoculate any bacteria into small size organisms such as Artemia could be a useful tool to study and understand the spread and life cycle of different bacteria related to Artemia and other small/micro crustaceans.

Necrotizing
hepatopancreatitis bacterium (NHPB) was selected to evaluate different inocula-tion procedures in Artemia sp.NHPB has caused the collapse of shrimp production in American and Latin American farms.The inoculation of NHPB into Artemia has been a difficult task in our laboratory.In addition, this bacterium has been successfully inoculated only into the penaeid shrimp (Gracia-Valenzuela et al., 2011).The aim of this experiment was to perform and evaluate different protocols to inoculate NHPB in Artemia sp.

MATERIALS AND METHODS
The protocols used to inoculate NHPB into Artemia were based on modifying the composition and concentration of bacterial inoculums.Three different procedures were evaluated; the experimental design consisted of triplicate tanks per treatment group.Each tank was a plastic container with 10 L of sterile and filtered marine water (salinity 35‰, temperature 25ºC, NH 3 -NH 4 0.01 mg•L -1 ) and 2 Artemia•mL -1 (35 day-old) were haphazardly stocked.Artemia were starved for 24 h prior to inoculation.
Necrotizing hepatopancreatitis bacteria was obtained from shrimp hepatopancreas sampled at commercial farms reporting the presence of the disease.The presence of the bacterium was confirmed by polymerase chain reaction and sequence analysis.Afterwards, the infected hepatopancreas were pooled and macerated to have homogeneous inoculums.The viability of the bacterium was confirmed by infecting healthy shrimp following the method described by Gracia-Valenzuela et al. (2011) and detecting the presence of NHPB after seven days.
In the first attempt (A), a NHPB inoculum was prepared, which consisted of macerated and homogenized shrimp hepatopancreas infected with NHPB (HP NHPB ), and mixed and homogenized with glycerol at a rate of 1:1 (w/v) (Ultra-Turrax T23; IKA Inc., USA).Afterwards, the inoculums were added and mixed into the water at a rate of 0.2 mL•L -1 .
The second attempt (B) was similar to the first procedure, but the inoculums were added at a rate 10 times higher (2 mL•L -1 ).For the third attempt (C), the HP NHPB +glycerol inoculum was mixed with formulated shrimp feed previously pulverized (Malta Cleyton ® ) at a ratio of 1:1 (v/w) and mixed into the water (0.2 mL•L -1 ).Controls for each treatment entailed adding similar inoculums but HPs free of NHPB were used.
Samples of Artemia were collected at 4 and 8 days post-inoculation and washed thoroughly with sterile marine water and distilled water, using a screen (0.5 mm mesh).After washing, Artemia was observed through a microscope (at 40 x magnification) to discard the presence of bacterial aggregates on the body.Artemia sub-samples were collected at four different locations within each experimental tank (10 organisms•location -1 ).
The presence of NHPB in Artemia was detected by the polymerase chain reaction.DNA was extracted using a commercial kit (GENECLEAN SPIN kit, Qbiogene ® ), and special primers were designed and used, considering the sequence U65509 GenBank: NHP/F2: 5'-CGT TGG AGG TTC GTC CTT CAG T-3' , NHP/R2: 5'-GCC ATG AGG ACC TGA CAT CAT C-3.Finally, PCR (PCR kit, Promega, Corp. USA) was performed under the following conditions: one cycle at 95ºC for 5 min, 35 cycles 94ºC for 1 min, 60ºC for 1 min, 72ºC for 1 min, and one final cycle at 72ºC for 10 min.Agarose gels (2%; E-Gel, Invitrogen Corp.) and UV transilluminator (Gel-Logic 100 Imaging System, KODAK) were further used to visualize the amplicons.The PCR products were purified using a purification kit (QIAquick ® ; QIAGEN, USA) and sent to a specialized laboratory (CISEI) to determine the nucleotide sequence.Thereafter, the sequences were compared to the sequence U65509 GenBank in the algorithm Blast N of the National Center for Biotechnology Information Bethesda, MD.

RESULTS AND DISCUSSION
The first two protocols (A and B) did not succeed to inoculate NHPB into Artemia; the organisms sampled 4 and 8 days post-inoculation were negative for NHPB.In contrast, Artemia was positive for NHPB in the third protocol; amplicons of 379-bp were observed in Artemia sampled on 4 th and 8 th days (Fig. 1).Th e bacteria were detected in 75% of the organisms on 4 th day, and in 100% on 8 th day.Th e control treatments of all the experimental protocols remained negative for NHPB during the trial.Th e amplicons sequences matched 100% with the NHPB reference sequence of GenBank (U65509).
Despite Artemia being starved, apparently they did not consumed the inoculated HP; it is possible that glycerol aff ected their attraction to HP.However, we tested pure and macerated HP NHPB as inoculums, and the results were also negative (unpublished data).Th us, these results suggest that it is diffi cult, if not impossible, to transfer NHPB from HP NHPB to Artemia, probably because the Artemia were not stimulated to consume the inoculum.It is possible that some experiments failed to demonstrate that Artemia was a vector of certain bacteria, or failed in trying to enrich Artemia with probiotic bacteria, because the bacteria did not enter through their digestive tract.Moreover, the inoculation of Artemia or other zooplankton species is commonly carried out through the exposure of the organisms to bacteria strains added to the water (Marques et al., 2005).Such a protocol may be useful in some experiments but not in others.
Th erefore, it is important in particular experiments to assure the consumption of inoculums by Artemia or the organism that is being evaluated.For instance, results from protocols A and B could conclude that Artemia cannot be inoculated by NHPB, but in protocol C the opposite was demonstrated.NHPB was detected only after four days from inoculation by protocol C, which indicates a rapid bacterial proliferation when they are attached to a substrate (formulated feed) consumed by Artemia.Vincent and Lotz (2005) demonstrated that shrimp exposed to HP NHPB resulted positive after 6-21 days; they observed that the bacterial spread was induced by the consumption of HP NHPB by experimental shrimp.Thus, the reason of the fast proliferation of NHPB could be attributed to a rapid consumption of the inoculums.In addition, the rapid consumption of the inoculums could be associated to the feeding attractants contained in the artificial feed; for instance, the formulation and addition of feeding attractants in shrimp feed were performed considering the chemical communication process in crustaceans (Barki et al., 2011).
Future experiments could contemplate the incorporation of artificial feed or feeding attractants for crustaceans in their inoculum formulation as a strategy to assure the consumption of any bacteria by Artemia or other zooplankton species.Such a protocol may avoid erroneous results.Moreover, this simple method could be useful in Artemia enrichment practices with probiotic bacteria.

Fig. 1 .
Fig. 1.Agarose gel electrophoresis analysis of PCR amplicons from extracted DNA from Artemia inoculated with NHPB by three different protocols.Protocol A: lanes 1-3, B: lanes 4-6, C: lanes 7-9.Th e positive NHPB control is shown in lane 10 (379-bp amplicon), the negative control in lane 12 and the low DNA mass ladder marker in lane 11 (Invitrogen).75% and 100% of sampled Artemia were positive four and eight days respectively aft er inoculation in the attempt using formulated feed.