Evaluation of survival and metamorphosis of larvae of Caligus rogercresseyi ( Boxshall and Bravo , 2000 ) ( Crustacea , Copepoda ) in Chile , depending on temperature , salinity and oxygen

The present study assessed the percentage of survival and metamorphosis of larval stages of Caligus rogercresseyi (Boxshall and Bravo, 2000) nauplius I, nauplius II to and nauplius II to copepodite, conducting bioassays in triplicate with 50 larvae each, nauplius I or nauplius II, at temperature of 12 °C, 15 °C and 18 °C; salinity 20, 23, 25, 2729, 31, 33 and 35 g/L and oxygen saturation with ranges between 30-60%, 90-100% and 190 200%. Bioassays were performed in station Quillaipe of Fundación Chile, Puerto Montt, Chile. It is concluded that the temperature is inversely proportional to the time of metamorphosis and survival of the larvae of Caligus rogercresseyi. In salinity is observed that increased this, greater is the percentage of survival and metamorphosis is faster, while the larvae do not survive less than 20g/L. Oxygen saturation ranges indicate that the larvae do not survive at saturations between 30-60%, and it had no differences between 90-100% saturation and 190-200%.


Introduction
Introduction farms, by being present in the marine environment are affected by ectoparasites on the Chilean native fauna, mainly Caligus rogercresseyi (Boxshall and Bravo, 2000;González et al., 2016).The life cycle of the parasite consists of three planktonic states, nauplius I, nauplius II, infective copepodite, and five parasitic stages, chalimus I-IV or juveniles and an adult (González and Carvajal, 2003).
Infective states feed on mucus, skin and blood of its host, causing focal and hemorrhagic ulcerative skin lesions, which can be of variable severity and injury of 2 to 5 millimeters in diameter to large ulcers on the body surface, also has the potential to affect growth, fertility, and the survival of their hosts (Boxshall and Bravo 2000;González et al., 2016).In addition to being a vector of bacterial and viral diseases, such ISA, IPN, SRS (Johnson et al., 2004;Oelckers et al., 2014).
According to information provided by Technological Institute of Salmon of Chile (INTESAL), carried out between the years 2000 to 2006, in the Lakes region (39-43° S), there was a decrease in the level of oxygen and an increase in mild in the salinity of the water, being these fundamental factors in the cycle of the parasite, in such a way that this situation in conjunction with other factors (wind speed, water depth, tides, circulation of water, currents, temperature and salinity patterns), productive practices and management could influence the increase of the levels of infestation by C. rogercresseyi in salmonid fish culture (Costello, 1993;Rozas & Asencio, 2007).The aim of this work was to evaluate the effect of temperature, salinity, and oxygen saturation, on survival, and metamorphosis of larvae under controlled conditions Caligus rogercresseyi.

Materials and Methods
Adults fish individuals of Salmo salar L., were infested by C. rogercresseyi, female's ovigerous, which were extracted manually using fine tweezers from previously anesthetized fish with "AQUÍ-S" (proportion 1:1000, active ingredient isoeugenol 50%).To provide an optimal environment, water filtered and sterilized sea was used by a UV intensity of 23800-25000uW/cm 2 with salinity of 31g/L, ambient temperature and constant aeration.
Specimens of C. rogercresseyi ovigerous females, were reared in 1 L beakers filled with seawater (31 g/L), each provided on average 50 larvae, the water was changed daily until 2100 larvae each bioassay to triplicate with a starting amount of 50 larvae.Every 3 hours the metamorphosis of nauplius I to nauplius II and nauplius II to copepod nauplius in all bioassays were counted until all metamorphosed or died.Counts and observations for a magnifier with 10x magnification was used, the larvae were collected with a sieve of 77 microns deposited in Petri dishes with 10 ml of water.The temperature was measured every 10 minutes using a Data -Logger, obtaining data with HOBO program.
Bioassay 1: Effect of temperature: three bioassays were conducted in triplicate at a constant temperature of 12 °C, 15 °C and 18 °C, precipitates prepared in 90 ml of water, with 31g/L salinity and aeration vessels constant.Kept in incubator refrigerator Velp model FTC 90E to maintain invariable temperature.
Bioassay 2: Effect of salinity: there were eight experiences at different salinities (20, 23, 25, 27, 29, 31, 33 and 35 g/L-1), vessels precipitated with 90 ml of water with aeration and constant temperature (15 °C).The vessels were stacked in an incubator Velp model FTC 90E, to maintain stable temperature.To obtain the expected salinity mixed freshwater with seawater or sea salt was added to obtain the desired salinity more accurately it was registered with the oxygen meter, salinity and temperature YSI model 85 -10 FT.
Bioassay 3: Effect of oxygen saturation: to determine the effect of oxygen saturation plastic trays with 15 L of water is used in a 40 ml container, with a salinity of 33 ppt.Each tray had oxygen and independent sensor diffuser to maintain saturation within the ranks of 30-60%, 90-100% and 190 -200%.
The analysis of the results included first a previous homocedasticity condition using Bartlet test, as previous condition to ANOVA, if it denotes significant differences it was applied Tukey test with a 95% confidence level.Determining if the difference between the evaluated groups is or not significant; P values less than 0.05 indicate significant differences.The SPSS15 program was used.

Bioassay 1: effect of temperature
Nauplius I at Nauplius II: at 18 °C they metamorphose took 12 hours at temperatures of 12 °C and 15 °C, it took 15 hours.Figure 1 shows that the highest percentage 12 °C metamorphosis, with 95% survival, followed by 15 °C (93%) and 18 °C (89%), the latter having not statistically significantly different temperatures 12 °C and 15 °C according to Table 1.
Nauplius II at Copepod: at 18 °C showed the shortest time of metamorphosis, 36 hours, 15°C followed by 42 hours and finally 12 °C taking 57 hours.According to Figure 2, at 12°C 91% of the nauplii II they reach their copepodite stage, showing a significant difference with 12 °C and 18 °C, see Table 2.
Nauplius II to Copepodite: the minumum time for copepodite was 42 hours at 3 g/L, 45 hours at 20, 31 and 35 g/L, 48 hours at 29g/L, 51 hours at 25 and 25 g/L and it late 54 hours at 23 g/L, being it the maximum time for reach metamorphosis between salinities (Figure 4).The maximum survival was 33 g/L, with 88% and following was 87% at 35 g/L, and the lower value was 20 g/L with 47%, existing significant differences at salinities analyzed (Table 4).

Discussion
The results revealed that for metamorphosis in both studied stages, and the survival is temperature dependent, and at high temperature, the metamorphosis time and survival decreased.These results agree with Tully and Whelan (1993), who mentioned that sea temperature increase is directly associated with short time parasite life cycle that in according to Bravo et al. (2009), the presence of low bodies females with low eggs chain.In according to Wootten et al. (1982), Johnson and Albright (1991), Tully and Whelan (1993), Boxaspen (1997), Wadsworth (1998), Pike and Wadsworth (1999), Bravo et al. (2013Bravo et al. ( , 2014) ) the growth rates, eggs production, survival and recruitment are directly associated to water temperature.Also, the life cycle development was affected by salinity and oxygen saturation.
The salinity effect accelerated the parasite life cycle, at high concentration, the survival increase, at 20 g/L the survival was low.Tucker et al. (2000) described that L. salmonis has high growth rate and recruitment at 34 g/L in comparison to 24 g/L salinity.The salinity does not retard the Caligus egg time production, but affect the larvae survival in analyzed studies, in according to Gonzalez and Carvajal (2003), salinities lower than 20 g/L does       not allow the survival of Nauplius and copepodite stages.It was showed that C. rogercresseyi does not survive at low oxygen concentration, and it reach 100% mortality between 30-60% in Nauplius II, and 97% in copepodite stages.At oxygen saturation upper than 90% it was not differences in time and survival.In according to the data base of Phytoplankton Monitooring Program of INTESAL done between 2000 to 2006, in Lakes region it was observed an oxygen decrease and a weak salinity increase, that are main factors in parasite life cycle, and in this scenario, these factors would affect the infestation rate by C. rogercresseyi (Rozas and Asencio, 2007;Bravo et al., 2014).

*
The mean difference is significant at 0.05.

Figure 2 .
Figure 2. Metamorphosis nauplii II to copepodite (lines) and mortality (bars), as a percentage, at different temperatures.

Figure 3 .
Figure 3. Metamorphosis nauplii I to nauplii II (lines) and mortality (bars), as a percentage, in terms of salinity.

Figure 5 .
Figure 5. Metamorphosis nauplii I to nauplii II (lines) and mortality (bars), as a percentage, different percentages of oxygen saturation.

Table 2 .
Statistical analysis of the survival in the metamorphosis of nauplii II to copepodite, at different temperatures.

Table 3 .
Statistical analysis of survival in the metamorphosis of nauplii I to II, at different salinities.
*The mean difference is significant at 0.05.

Table 4 .
Statistical analysis of survival in the metamorphosis of nauplii II to copepodite, in terms of salinity.
I (Salinity) g/L J (Salinity) g/L P I (Salinity) g/L J (Salinity) g/L *The mean difference is significant at 0.05.