4.0 Establishment of a new C. sancticaroli cultures: difficulties and pathways
Before starting a new culture of C. sancticaroli, it is necessary to prepare all the materials that are going to be used (Table 2) and prepare a temperature and photoperiod controlled environment where the culture will be maintained. It is important that all the materials are exclusively used for the culture in order to avoid contamination.
Table 2
Materials needed to start one C. sancticaroli tray.
Item | Quantity |
Sediment | 1.5 kg |
Culture water | 4 liters |
Tray | 1 unity |
Cage | 1 unity |
Fish food* | 0.005g/ml stock solution (75 ml/week) |
Air pump | 1 unity |
Algae solution* | 10 ml |
Tweezers | 1 unity |
Beakers | 1 unity |
Blender | 1 unity |
*In our cultures we have used Tetramin® fish food and Raphidocelis subcapitata algae cultures as food resource. |
C. sancticaroli organisms were obtained from the Ecotoxicology Laboratory of the Center for Water Resources and Applied Ecology, at São Carlos School of Engineering, from the University of São Paulo (São Carlos, Brazil). Five ovigerous masses were brought to the Plankton Ecology Laboratory at the Federal University of Juiz de Fora (Juiz de Fora, Brazil) for the establishment of new cultures starting with methods described by Fonseca and Rocha (2004). The ovigerous mass were kept in a 100 ml beaker and were fed with 10 ml of Raphidocelis subcapitata (2193916,67 cells/mL; Da costa et al., 2014; Barbosa et al., 2019). The water used was purchased with adequate characteristics for a good C. sancticaroli survival (Table 3).
Table 3
Characteristics of the water used for C. sancticaroli cultivate.
Parameter | Value |
pH | 7.28 (25ºC) |
Electric conductivity | 197 µS/cm |
Calcium | 4.22 mg/L |
Magnesium | 0.999 mg/L |
Bicarbonate | 108.32 mg/L |
Phosphate | 0.51 mg/L |
Sodium | 38.299 mg/L |
Sulfate | 7.32 mg/L |
Chloride | 2.38 mg/L |
In the beginning, the number of individuals hatching from the ovigerous mass in our laboratory culture ranged from 150 to 250 individuals. However, the study of Fonseca and Rocha (2004) reported that hatching around 500 to 600 individuals of the ovigerous mass. This difference was probably due to the manipulation during the collection of the mass on the trays and its transport to the beakers, or due to the amount of algae solution added, as many algae became entangled and prevented it from hatching completely. We used tweezers to collect the ovigerous mass very carefully, and the algae solution was added in the beaker only when the 1st instar larvae hatched. With these small precautions we verified an increase of survival larvae.
Up to 48 h, the water was kept static (no aeration) and all the masses hatched. After 48 h, the hatched larvae (1 st instar) were transferred to 7-liter plastic trays (45.5 x 28 x 7.7 cm), containing 1/3 of sediment (river gravel granulometry 03) and 2/3 of water (about 4 liters; characteristics on Table 3). A cage was adapted in the top of the tray to keep unwanted organisms out of the culture and to keep the newly hatched adults in contact for mating and reproduction (Fonseca and Rocha, 2004). The cultivation water had a pH between 6.5 and 7.5, the culture room conditions consisted of a temperature range of 22–25°C and a 12h light:12h dark photoperiod. The culturing trays were continuously aerated with air pumps (Fig. 4). After 2 days of cultures establishment (without food), the larvae were fed with 25 ml Tetramin® fish food solution (0.005 g/ml stock solution) three times a week. Such feeding type was done for all stages of the instar, we have not tested applying different concentrations of food for different stages; this may be a suggested analysis for future studies. Water needs to be replenished frequently (once/week; approximately 34% renewal), until it reaches the initial volume of the tray.
In ecotoxicological tests, it is important to establish the number of larvae that will be used per treatment. Based on the articles we found, there is not a consensus regarding the number of larvae used neither acute nor chronic tests using C. sancticaroli, although we noticed that 6 larvae per treatment was commonly used in acute tests (Campagna et al., 2013; Colombo-Corbi et al., 2017; Dornfeld et al., 2019; Guimaraes-Souto et al., 2018; Janke et al., 2011; Novelli et al., 2012; Silvério et al., 2005; Yamada et al., 2012)
The first generation of new ovigerous masses was obtained 1 month after the establishment of the initial tray. After that, three masses from the main tray were removed and placed in a beaker filled with culture water and 10 ml of the Raphidocelis subcapitata algae solution. After hatching, the larvae of the 1 st instar were placed in a tray as described above (300 organisms/tray). This procedure was repeated until the new culture had enough trays for a test (approximately 4 or 5 trays).
4.1 General care
When food solution or water are added to the tray, care must be taken to not revolve the sediment, stressing the larvae (Fig. 5.A). When removing the cages to maintenance, it is important to tap the sides of the cage gently, so that mosquitoes fly upwards and do not leave the cage (Fig. 5.B). Additionally, the pumps aeration must be soft, without revolving the sediment.
4.2 Feeding the organisms
As commented above, we have used Tetramin® solution to feeding the organisms. Most studies also used a solution of Tetramin® (Silva et al., 2019; Yamada et al., 2012; Sotero-Santo et al., 2017, Morais et al., 2020) or another fish food, such as Nutrafish® or Dog Chow® ration (Beguelli et al., 2018; Morais et al., 2014; Palacio-Cortes et al., 201), but information on methods for the preparation and stocking it are insufficient in the literature. We beat 2.5 g of Tetramin® solution and 500 ml of distilled water in a blender. After prepared, the stock solution must be stored in the refrigerator at 4°C (there was no need to freeze) to avoid the fermentation and it is important to remove the volume of solution that is going to be used 20 to 30 minutes before feeding the larvae or until it is at room temperature. This solution usually lasts for 1 month in the refrigerator.
4.3 Temperature of the culture room
Temperature is an important parameter for the development and survival of macroinvertebrate species in the laboratory (Strixino & Strixino, 1985). We have observed this, when culture room was colder, consequently, life cycle was delayed, also delaying the start of tests. Therefore, any problem with controlling room temperature must be resolved quickly in order to not delay or accelerate C. sancticaroli development and survival. Our room temperature ranged between 22°C and 25°C and many studies maintain the culture in the same condition (see supplementary material Table S1), but it is important that each cultivation room has stabilized temperatures.
4.4 Cleaning the trays and the room
The culture and maintenance of the trays are extremely important and must be made with care to guarantee the larvae survival. The manipulation must be minimal, only to provide the essential, and all the materials used must be washed correctly (alkaline detergent, 1h-bath of 10–20% of HCl solution, and then a rinse with distilled water) to avoid contamination.
In one of our experiments we observed the existence of fungus in some of the trays, which in our case probably came from building wall where the experiment was performed or probable by overfeeding the trays, but brief suspension of feeding was not tested. Experiments taking into account different amounts of food for chironomid life stages are suggested.
In addition to the correct washing of materials and the tray, it is important that the culturing room be also cleaned constantly, including walls and, even the number of people who enter the place should be limited, requiring that everyone responsible for maintenance are equipped with the correct I.P.E (Individual Protection Equipment) when entering the room (coat, gloves, and hair up). Taking these precautions ensures the health of the culture.