Computational protocol: Alona iheringula Sinev & Kotov, 2004 (Crustacea, Anomopoda, Chydoridae, Aloninae): Life Cycle and DNA Barcode with Implications for the Taxonomy of the Aloninae Subfamily

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Protocol publication

[…] Some females were collected in the littoral region of a dammed portion of the Cabo Verde River/Furnas Reservoir (21° 26′05″ S, 46° 10′57″ W), in the southern region of Minas Gerais State, Brazil, by vertical and horizontal hauls using a zooplankton net of 68 µm mesh size. It is a public area and no special permission is necessary to authorize access. The species involved are not endangered or protected. Therefore, there are no restrictions on collecting. The sample was collected near macrophytes in February 2009.In the laboratory, parthenogenetic females of Alona iheringula () were isolated and placed in 2 L beakers containing reconstituted water, according to ABNT . The culture media and food suspension were renewed every two days. Specimens were acclimated for about 10 generations (30 days). The pH of the culture media was 7.4; the electric conductivity was 180 µ−1 and the hardness was 46 mg.L−1 CaCO3. Experimental cultures were maintained in germination chambers with a photoperiod of 12h-light/12h-dark and temperature of 25(±1)°C (a mean temperature observed in the sampling area when the organisms were collected). The organisms were fed with Pseudokirchneriella subcapitata algae, cultured in Chu 12 medium and cropped in the exponential phase, with 105 cells per individual and a suspension of mixed suspension (fish-food and biological yeast), supplied in equal proportions , .Twenty females were isolated and maintained until the production of neonates. The 40 neonates of less than 24 hours old were placed in 100 mL of culture medium in polypropylene bottles and kept in a germination chamber with temperature, light and feeding conditions as specified above. The isolated organisms were observed two or three times a day under the stereoscopic microscope to obtain life cycle data (development times, longevity and fecundity). Individual body growth was measured daily under optical microscope using a micrometric grid and 40× magnification.In the same collecting coordinates, qualitative samples of the zooplankton community were collected (near the macrophytes) using a net of 68 µm mesh size, every two months, from June 2008 to October 2009. The samples were fixed in formalin 4% and kept in the Coleção do Laboratório de Limnologia (sample CL3053), at the Universidade Federal de Alfenas, MG (UNIFAL-MG). From Secchi disk and chlorophyll-a data, obtained from the same place, the Trophic State Index was calculated by Carlson methodology, adapted by Toledo et al . The chlorophyll concentration was determined using the extraction method with 90% acetone as described in Golterman et al .For the DNA barcode analysis, the specimens were fixed with 90% EtOH and placed into pure water for 12 h for cleaning. Genomic DNA was extracted using phenol extraction and ethanol precipitation . To amplify the mitochondrial COI gene, the internal primers Chy-f 5′-TTG GGG ATG ATC AAATTT ATA ATG T-3′ and Chy-r 5′-AGA GGT ATT CAG ATT TCG ATC TGT CA-3′ were used. PCR reactions had a total volume of 25 µL and were performed according to Ivanova et al using Platinum Taq (Invitrogen) as the enzyme. The PCR conditions were 94°C for 3 min as initial denaturation and 40 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 90 s. Direct DNA sequencing was done using purified Exo-SAP (Fermentas) PCR products, carried out in a 3130xl Genetic Analyzer (Life Technologies, Carlsbad/CA/USA) automated DNA sequencer, following the manufacturer's instructions. The sequences were obtained bidirectionally for accurate reading (Genbank access number KF383284).The Alona iheringula COI smaller fragment (using Chy-f and Chy-r, ) was aligned in MEGA 5 with other Aloninae COI sequences obtained from Barcode of Life Data Systems (BOLD) (, ). The Kimura 2-Parameter (K2P) distance model was used to calculate sequence divergences and the GTR+G+I was found to be the best substitution model obtained for the alignment (+G, parameter  =  2.7514;[+I], 60.4423% sites). Identification trees were generated by MEGA 5 facilities. Nonparametric bootstrapping was performed using 1000 replicates. […]

Pipeline specifications

Software tools MEGA, BOLD
Application WGS analysis
Organisms Saccharomyces cerevisiae