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Biomass estimation of the terrestrial ecotoxicological species Folsomia candida (Collembola) using a real-time polymerase chain reaction
Ecotoxicology and Environmental Safety 101 (2014) 59–63
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Biomass estimation of the terrestrial ecotoxicological species Folsomia candida (Collembola) using a real-time polymerase chain reaction Li-Bo Hou a,n, Yukinari Yanagisawa a, Shunji Yachi b, Nobuhiro Kaneko a, Taizo Nakamori a a b
Laboratory of Soil Ecology, Graduate School of Environment and Information Sciences, Yokohama National University, Japan National Institute for Agro-Environmental Sciences Organo-Chemicals Division, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 19 August 2013 Received in revised form 4 December 2013 Accepted 18 December 2013 Available online 11 January 2014
The abundance and growth of the Folsomia candida soil arthropod have been widely used to assess the environmental impact of a range of soil pollutants, and increasing concerns about environmental pollution require advanced and rapid methods to estimate ecological toxicity. Here, we developed a quantitative polymerase chain reaction (qPCR)-based assay for determining the biomass of F. candida. Prior to DNA extraction, an appropriate amount of an artificial sequence was spiked into the test samples, allowing us to assess the extraction efficiency used for normalisation. We designed primers based on the sequencing information of the nuclear RNA polymerase II (Pol II) and mitochondrial cytochrome c oxidase subunit I (mtCOI) genes of F. candida. Assays were performed on samples containing a different number of individuals at the same body length (individually same biomass; same age) and samples containing the same number of individuals at a different body length (individually different biomass; different age). Biomass was estimated from the body lengths of collembolan samples. For both genes, DNA quantity showed a significant linear relationship between increased collembolan numbers and the estimated biomass; DNA quantity in different ages of collembolans showed a significant correlation with body length and a linear relationship with the estimated biomass. We believe that this rapid and accurate technique could be used to detect and quantify soil animals and thus would improve ecotoxicological testing. & 2013 Elsevier Inc. All rights reserved.
Keywords: Body length Dry weight DNA quantity Ecotoxicity test Number qPCR
1. Introduction In terrestrial ecosystems, many soil animals are sensitive to environmental pollutants. In ecotoxicological tests of chemicals on organisms, several different endpoints are widely used, including survival, growth and reproduction, to determine the impact of toxicity on soil animals (van Gestel, 2012). The biomass is one of the indices that represent the importance in ecosystem function (Gruner, 2003) and is related to traditional endpoints, such as growth and survival. The collembolan, Folsomia candida, has commonly been used in ecotoxicological tests (Fountain and Hopkin, 2005; Natal-da-Luz et al., 2008) and is considered the “standard” test species by the International Organization for Standardization (ISO, 1999) and the Organization for Economic Co-operation & Development (OECD, 2009). For F. candida, a direct measurement of biomass is often difficult due to their minute size (Caballero et al., 2004). Thus, researchers have published relatively easier, more reliable methodologies for measurement, such as length to biomass function (Ganihar, 1997; Caballero et al., 2004). However, body length measurement of a large amount of small animals is time-consuming work. Increasing concern about n Correspondence to: Laboratory of Soil Ecology, Graduate School of Environment and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan. Fax: þ 81 45 339 4379. E-mail address: [email protected] (L.-B. Hou).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.12.011
environmental pollution requires advanced and rapid methods to estimate ecological toxicity (Nota et al., 2008). The quantification of DNA using real-time polymerase chain reaction (PCR) is a fast, effective and accurate method and is applicable to small amounts of sample (Lwin et al., 2011). The method has been applied to some soil animals (nematodes: Berry et al., 2008) but not yet to collembolans. The quantification of DNA in a sample is subject to inaccuracies caused by losses during DNA extraction or inhibition by co-extracted compounds. Spiking the sample with a unique (alien) nucleic acid allows for the assessment of extraction efficiency, and this value can be used for normalisation. This method benefits from the fact that an accurately defined amount of the spike can be introduced prior to extraction, permitting good estimation of the error that is introduced through most stages of processing (Dorak, 2006). Daniell et al. (2012) have demonstrated that an artificial spike to samples allows both a significant improvement in the estimation of DNA quantity and a decrease in the variability in the estimation. The aim of the current study was to develop a quantitative PCR (qPCR)-based assay for determining the biomass and number of F. candida. We conducted two experiments, one to assess the relationship between DNA quantity and collembolan number, and the other, to determine the relationship between DNA quantity and body length. The genes, nuclear RNA polymerase II (Pol II) and mitochondrial cytochrome c oxidase subunit I (mtCOI) were selected for estimation. The mtCOI gene is frequently used in
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phylogenetic studies of animals; in insects, the Pol II gene is believed to be a single-copy gene (Wiegmann et al., 2009), thus giving more precise measurements of DNA copy number (Tellenbach et al., 2010). DNA fragments of an artificial sequence were spiked into the samples to serve as the one constant control for the experimental errors and losses that occur during DNA extraction. Assays were performed on samples containing different numbers of individuals at the same body length (same biomass) and samples containing the same number of individuals at a different body length (different biomass). Biomass was estimated from the body length.
2. Materials and methods 2.1. Test organisms and culture conditions Animals for testing were obtained from laboratory stock cultures of F. candida from a population in central Japan (Itoh et al., 1995) and reared on baker0 s yeast, as described by Nakamori et al. (2008). Our laboratory population represented the offspring of a single individual. Age-synchronised collembolan specimens were prepared according to the ISO (1999). Specimens were collected in 1.5-ml tubes, preserved in 100 percent ethanol, and stored at 20 1C until use.
2.2. Experimental design For DNA quantity–number experiments, different numbers of collembolans (1, 5, 10, 20, 30, 40 and 50 individuals) at 13–16 and 34–37 days of age were collected, and two replicates for each number of individuals at each age were performed; for DNA quantity–body length experiments, 10 specimens with two replicates were collected at each age (0–3, 6–9, 13–16, 20–23, 27–30 and 34–37 days of age). To clarify the relationship between collembolan biomass and an increase in DNA quantity, age-related changes in body length were used as the parameter for assessing collembolan growth.
2.3. Measurements of body length Before DNA extraction, the length of the collembolans from mouthpart to anus was measured, placing the specimens side down in ethanol under a digital microscope, VHX-1000 (Keyence corporation, Osaka, Japan). For the different numbers of samples at the same ages, measurements were performed on 20 specimens for each age, and the mean value was used; for the body length measurements at different ages, 10 individuals with two replicates were measured.
2.4. Biomass estimation The regression between F. candida body length and dry weight was calculated assuming a power function, W ¼6.457L2.990, where L is the linear length (mm) and W is the weight (mg) (Caballero et al., 2004; Tanaka, 1970).
2.5. Sequencing The sequences of the Pol II and mtCOI genes were determined for designing PCR primers. Our population consisted of the offspring of a single individual (established in 2006). Genomic DNA was extracted from eggs using a QIAGEN DNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer0 s instructions. The parent specimen was preserved in the Tottori Prefectural Museum, Japan (No. TRPM-AAr0000630). The DNA sequence comprising a 658-bp region of the mtCOI gene was obtained following Protocol 1 reported by Nakamori (2013). For the Pol II gene, the 588-bp DNA sequence was determined in the same manner but using different primer pairs and PCR conditions. The primer pair used was Fc-PoIIGF (50 -GTCCGTCATGGTTCACTACG-30 ) and Fc-PoII-GR (50 -CTGCTGGAACCTCGTCTCTA-30 ). The reaction condition included an initial denaturation step at 95 1C for 10 min followed by 35 cycles at 95 1C for 15 s and 60 1C for 1 min with a final annealing/extension step at 60 1C for 7 min. All sequences were verified as originating from collembolans using the BLASTn algorithm of the basic local alignment search tool against GenBank. The sequences were submitted to GenBank (Accession Nos. AB845557 for the mtCOI gene; AB845285 for the Pol II gene).
2.6. Spike-in-control-preparation The PCR products from an artificial DNA sequence were used as a spike-incontrol. One artificial sequence was designed (Table 1). The DNA fragment was synthesised by GenScript USA Inc. The 83-bp DNA fragment was amplified using the ArtL2 (50 -CTTGAAGCGACCGTTACACA-30 ) and ArtR2 (50 -CGTAATTGGCCTCAGATCGT-30 ) primers. Amplification was performed in 25-ml reaction volumes containing 1 ml of DNA template, 12.5 ml of AmpliTaq Golden MasterMix (Applied Biosystems), and 25 mM of each primer. The reaction condition included an initial denaturation step at 95 1C for 10 min followed by 35 cycles at 95 1C for 15 s and 60 1C for 1 min with a final annealing/extension step at 60 1C for 7 min. The PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN), eluted with pure water and stored at 20 1C. The amplicons from the artificial sequence (20 ml, 5 10 4 ng/ml) were spiked in the test samples prior to DNA extraction. The PCR amplicons were also used for generating calibration curves in real-time PCR. The artificial sequence cannot be found in the sequences of the test samples.
2.7. DNA extraction Ethanol was removed from the screw cap microtubes (2-ml) by pipette, and the residual volume was air-dried for 10 min. Collembolan DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN) with some modifications. Sterile glass beads (0.1 mm in diameter, 0.2 g and 2.50–3.50 mm in diameter, number 8) were added to the tubes containing the samples; ATL buffer (270 ml) was added, and the contents were mixed well and placed on a shaker for 10 min at 30 Hz (1/s) frequency using a QIAGEN Tissue Lyser II. Samples were frozen for 20 min at 70 1C and thawed at room temperature, and then, the process was repeated. Thirty microlitres of proteinase K was added, and the contents were mixed well by vortexing and incubated at 56 1C overnight with rotation at 100 rpm/min in a hybrid oven. Further extraction procedures used 200 ml of mixture and followed the manufacturer0 s instructions (QIAGEN). The purified DNA was eluted into 80 ml of pure water and stored at 20 1C until use.
2.8. qPCR qPCR experiments were performed in 0.2-ml 96 well plates using the StepOnePlus Real-time PCR system (Applied Biosystems). Each reaction contained 0.95 ml of DNA as the template, 3.8 ml of water, 0.625 ml of each 100 mM forward and reverse primer (CF1, 50 -AACAACGCCACGCTTCTATT-30 and CR1, 50 - GTCGACAGGCGAAATTCTTC-30 for Pol II gene; FcCOI-A-F 50 -GGGTATGACTTGGGATCGAA-30 and FcCOI-A-R 50 -CTCCAGCAAGAACTGGAAGG-30 for mtCOI gene; ArtL2 and ArtR2 for spike-in-control), 6.25 ml of SYBR master mix and 0.25 ml of 50ROX (TOYOBO THUNDERBIRD SYBR qPCR Mix) in a final volume of 12.5 ml. The cycling parameters were as follows: an initial denaturation step at 95 1C for 10 min followed by 40 cycles at 95 1C for 15 s and 60 1C for 1 min, with a final annealing/extension step at 60 1C for 10 min. DNA melting curve analysis was performed. The primers were designed using Primer3 software (Rozen and Skaletsky, 2000). Standard curves were constructed in every run to calculate the concentrations of unknown relative to standard samples. The PCR products obtained using the qPCR primers were used as the standard samples. The DNA from the eggs of F. candida was extracted using the DNeasy Blood & Tissue kit (QIAGEN) and amplified using the AmpliTaq Gold Master Mix (Applied Biosystems), according to the manufacturer0 s instructions. The PCR conditions were the same as those used in the preparation of the spike-in-control, as described above. Standard curves were obtained in a duplicate series of six 10-fold dilutions of standard samples. Each run contained unknowns and standards. The PCR reactions were performed in technical duplicate for unknowns, and mean values were used. The data were normalised to the DNA quantity in the spike-in-control. Normalised values of all the samples were expressed relative to the mean normalised values of a reference treatment. For DNA quantity–number experiments, treatment with a single 13–16-day-old collembolan was used as the reference treatment; for DNA quantity–body length experiments, treatment with a 0–3-day-old collembolan was used as the reference treatment.
2.9. Data analysis Data were analysed using the R software (R Development Core Team, 2011). The relationships between DNA quantity–number and DNA quantity–dry weight were analysed using the linear regression formula, y¼ ax, where a is the constant, x is the number of individuals or dry weight (mg), and y is the DNA quantity. Curves for the DNA quantity–body length relationship were fitted to the data using the non-linear regression module of the R software. The formula was as follows: y¼ ax3, where a is the constant, x is the body length (mm) and y is the relative DNA quantity. The relationship of the relative ratio of mtCOI/Pol II–age was analysed based on the average data.
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3. Results
4. Discussion
3.1. DNA quantity–number
We developed a qPCR-based assay for determining the biomass of the collembolan, F. candida. We believe that this technique could be used to detect and quantify soil animals and would thus improve ecotoxicological testing. Although the qPCR-based assay represents a rapid and accurate way of estimating biomass, attention should be paid to the target genes and samples that could possibly influence the accuracy of the estimation. The biomass estimation was possible using both nuclear and mitochondrial genes, Pol II and mtCOI. Pol II gene was expected to be a good indicator of biomass because the number of nuclear genes per cell cannot change through a lifetime in animals. On the other hand, the results of the mtCOI gene assay should be interpreted with
Significantly positive linear correlations were observed between relative DNA quantity and collembolan number for the Pol II and mtCOI genes (Fig. 1; linear model, Po0.001). 3.2. DNA quantity–body length The attained cubic regression equations of relative DNA quantity to collembolan body length (mm) were y¼ 3.9024x3 (Pol II gene) and y ¼7.5167x3 (mtCOI gene; non-linear regression analysis: all the parameters of the non-linear model were significant at P o0.001; Fig. 2). 3.3. DNA quantity–biomass Significantly positive linear correlations were observed between relative DNA quantity and the dry weight of collembolan samples in each gene assay (Pol II and mtCOI; Figs. 3 and 4; linear model, Po0.001). The equations of the regression lines at different ages were slightly different (Fig. 3); using relative DNA quantity as the variable (y) and collembolan biomass (mg) as the predictor variable (x); the values were y¼0.06x (Pol II gene) and y¼0.115x (mtCOI gene). In the experiments with different numbers of individuals, 13–16-day-old and 34–37-day-old collembolans showed different slopes of regression lines (Fig. 4; linear model, P o0.001). 3.4. mtCOI/pol II The relative ratio of the mtCOI/Pol II genes changed with age (Fig. 5). The ratio was increased with age, especially at 27–30 days of age.
Fig. 2. The relationships between relative DNA quantity and collembolan body length, as measured by mitochondrial cytochrome c oxidase subunit I (mtCOI) and nuclear RNA polymerase II (Pol II) gene assays. Solid dots with a solid line represent the Pol II gene assay; hollow dots with a dashed line indicate the mtCOI gene assay; y¼3.9024x3 (Pol II gene) and y¼7.5167x3 (mtCOI gene). Each dot contains DNA from 10 collembolan individuals.
Fig. 1. The relationships between relative DNA quantity and collembolan number, as measured by mitochondrial cytochrome c oxidase subunit I (mtCOI) and nuclear RNA polymerase II (Pol II) gene assays. Solid dots with a solid line indicate 13–16-day-old collembolans; hollow dots with a dashed line represent 34–37-day-old collembolans. y¼ 1.752x (Pol II gene, 13–16 days old; R2 ¼0.9866); y¼ 7.753x (Pol II gene, 34–37 days old; R2 ¼ 0.9820); y¼2.315x (mtCOI gene at 13–16 days old; R2 ¼0.9883); y¼ 23.417x (mtCOI gene, 34–37 days of age; R2 ¼ 0.9757); Po 0.001 for all.
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caution, as mitochondrial number per cell and/or the number of mitochondrial genome per mitochondrion can change with physiological status; thus, the use of a mitochondrial gene may result in an incorrect estimation of biomass. It is known that increased mitochondrial number and mass result from the growth and division of preexisting organelles (Seo et al., 2010); mitochondrial DNA copy number is closely tied to reproduction (Lemire, 2005). In fact, the ratio of the mtCOI and Pol II genes had some variance with the growth of collembolans, but this variance was negligible for the biomass estimation in the present study. Although cautious data interpretation is needed, the mtCOI gene assays will be increasingly applicable to species identification and abundance estimation for other species.
Fig. 3. Correlations between collembolan dry weight and relative DNA quantity (same number of individuals; different ages). The regression lines were based on the calculation of body length to dry weight. Body lengths were measured at ages ranging from 0–3 to 34–37 days old. Solid dots with a solid line represent the nuclear RNA polymerase II (Pol II) gene assay; hollow dots with a dotted line indicate the mitochondrial cytochrome c oxidase subunit I (mtCOI) gene assay; y¼ 0.06x (Pol II gene) and y ¼0.115x (mtCOI gene). Each dot contains DNA from 10 collembolan individuals.
Because the mtCOI gene has been proposed as the standard barcode for animals, more and more sequence data for the gene are becoming available in public databases (Valentini et al., 2009). Egg production in the collembolan samples might cause variation in DNA quantity at 27–30 days of age. F. candida is a parthenogenetic species, and the animals become sexually mature at 21 and 24 days of age at 20 1C (Fountain and Hopkin, 2005). Around these ages, the samples before and after egg production would have different DNA quantities. Similar phenomena may occur in sexual species. Therefore, for the application of DNAbased biomass quantification methods to sexual species, further studies are required. We hypothesised that there would be differences in the DNA quantity and corresponding dry weight relationships between collembolan juvenile (13–16-day-old) and adult (34–37-day-old)
Fig. 5. The ratio of the DNA quantity from the mitochondrial cytochrome c oxidase subunit I (mtCOI) relative to the RNA polymerase II (Pol II) gene assay. Each dot contains DNA from 10 collembolan individuals.
Fig. 4. Correlations between collembolan dry weight (individual numbers: 0–50) and relative DNA quantity (different number of individuals; same age), as measured by mitochondrial cytochrome c oxidase subunit I (mtCOI) and nuclear RNA polymerase II (Pol II) gene assays. Relative DNA quantity was regressed against collembolan dry weight for each gene. Solid dots with a solid line indicate 13–16-day-old collembolans; hollow dots with a dashed line represent 34–37-day-old collembolans. y¼ 0.423x (Pol II gene, 13–16 days old; R2 ¼ 0.9705); y¼0.100x (Pol II gene, 34–37 days old; R2 ¼0.9444); y ¼0.563x (mtCOI gene, 13–16 days old; R2 ¼0.9775); y¼ 0.298x (mtCOI gene, 34–37 days old; R2 ¼ 0.9208); Po 0.001 for all.
L.-B. Hou et al. / Ecotoxicology and Environmental Safety 101 (2014) 59–63
animals because of the changes in both cell size and cell mass with animal age. Studies in rats have suggested the partition of normal growth into three periods as follows: cell division alone, cell division with concomitant cell enlargement and cell enlargement alone with no further increase in the number of cells (Winick and Noble, 1965). Detection of differences in number can be applied to survival and avoidance tests (Natal-da-Luz et al., 2008), and the detection of differences in body length and estimated biomass can be applied to growth tests. Absolute quantification can also be used in these ecotoxicity tests, but this type of quantification requires calibration curves generated from known samples. Preparing a sample series of known numbers, biomass, and/or body length needs further study. The regression equations (DNA quantity vs. number, body length, and/or biomass) estimated in the present study could not be used directly if the sample preparation, DNA extraction, and/or PCR conditions differed in each occasion and laboratory. Our DNA quantification methods in combination with RNA analysis will help link the traditional endpoints with transcriptional responses in ecotoxicology tests. Transcriptional analysis has received increasing attention and became popular in ecotoxicology, but there is still a considerable gap between an understanding of biological effects and transcriptional responses (van Gestel, 2012; van Straalen and Roelofs, 2008). DNA can be easily co-extracted with RNA, allowing for the simultaneous analysis that would bridge the gap. In the future, molecular-based techniques from a genomic level, particularly qPCR, to predict the biomass of soil animals will be widely used. The application of our method could be huge benefit to soil studies; qPCR-based biomass estimations may not only serve as an endpoint but it could also aid in determining endpoints (survival, mobility, reproduction and respiration) for toxicity assessment. The design of species-specific primers will allow this technique to be applied to mixed collembolan species cultures in the future.
5. Conclusion In conclusion, biomass estimation was possible with both the nuclear Pol II and mtCOI gene assays. Although the quantity of the mtCOI gene was variable, it can still be used for the biomass estimation of soil animals. We believe that this technique would, thus, improve ecotoxicological testing.
Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research (No. 24241014; Project leader: Dr. M. Kamo) from the Ministry of Education, Culture, Sports, Science and Technology, Japan; and a grant from Yokohama National University and the Sasakawa Scientific Research Grant from The Japan Science Society. We are grateful to Dr. K. Ichisawa for archiving our specimen in the Tottori Prefectural Museum of Japan. The authors would like to thank T. Otaki at Yokohama National University for the support of DNA sequencing.
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Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Biomass estimation of the terrestrial ecotoxicological species Folsomia candida (Collembola) using a real-time polymerase chain reaction Li-Bo Hou a,n, Yukinari Yanagisawa a, Shunji Yachi b, Nobuhiro Kaneko a, Taizo Nakamori a a b
Laboratory of Soil Ecology, Graduate School of Environment and Information Sciences, Yokohama National University, Japan National Institute for Agro-Environmental Sciences Organo-Chemicals Division, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 19 August 2013 Received in revised form 4 December 2013 Accepted 18 December 2013 Available online 11 January 2014
The abundance and growth of the Folsomia candida soil arthropod have been widely used to assess the environmental impact of a range of soil pollutants, and increasing concerns about environmental pollution require advanced and rapid methods to estimate ecological toxicity. Here, we developed a quantitative polymerase chain reaction (qPCR)-based assay for determining the biomass of F. candida. Prior to DNA extraction, an appropriate amount of an artificial sequence was spiked into the test samples, allowing us to assess the extraction efficiency used for normalisation. We designed primers based on the sequencing information of the nuclear RNA polymerase II (Pol II) and mitochondrial cytochrome c oxidase subunit I (mtCOI) genes of F. candida. Assays were performed on samples containing a different number of individuals at the same body length (individually same biomass; same age) and samples containing the same number of individuals at a different body length (individually different biomass; different age). Biomass was estimated from the body lengths of collembolan samples. For both genes, DNA quantity showed a significant linear relationship between increased collembolan numbers and the estimated biomass; DNA quantity in different ages of collembolans showed a significant correlation with body length and a linear relationship with the estimated biomass. We believe that this rapid and accurate technique could be used to detect and quantify soil animals and thus would improve ecotoxicological testing. & 2013 Elsevier Inc. All rights reserved.
Keywords: Body length Dry weight DNA quantity Ecotoxicity test Number qPCR
1. Introduction In terrestrial ecosystems, many soil animals are sensitive to environmental pollutants. In ecotoxicological tests of chemicals on organisms, several different endpoints are widely used, including survival, growth and reproduction, to determine the impact of toxicity on soil animals (van Gestel, 2012). The biomass is one of the indices that represent the importance in ecosystem function (Gruner, 2003) and is related to traditional endpoints, such as growth and survival. The collembolan, Folsomia candida, has commonly been used in ecotoxicological tests (Fountain and Hopkin, 2005; Natal-da-Luz et al., 2008) and is considered the “standard” test species by the International Organization for Standardization (ISO, 1999) and the Organization for Economic Co-operation & Development (OECD, 2009). For F. candida, a direct measurement of biomass is often difficult due to their minute size (Caballero et al., 2004). Thus, researchers have published relatively easier, more reliable methodologies for measurement, such as length to biomass function (Ganihar, 1997; Caballero et al., 2004). However, body length measurement of a large amount of small animals is time-consuming work. Increasing concern about n Correspondence to: Laboratory of Soil Ecology, Graduate School of Environment and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan. Fax: þ 81 45 339 4379. E-mail address: [email protected] (L.-B. Hou).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.12.011
environmental pollution requires advanced and rapid methods to estimate ecological toxicity (Nota et al., 2008). The quantification of DNA using real-time polymerase chain reaction (PCR) is a fast, effective and accurate method and is applicable to small amounts of sample (Lwin et al., 2011). The method has been applied to some soil animals (nematodes: Berry et al., 2008) but not yet to collembolans. The quantification of DNA in a sample is subject to inaccuracies caused by losses during DNA extraction or inhibition by co-extracted compounds. Spiking the sample with a unique (alien) nucleic acid allows for the assessment of extraction efficiency, and this value can be used for normalisation. This method benefits from the fact that an accurately defined amount of the spike can be introduced prior to extraction, permitting good estimation of the error that is introduced through most stages of processing (Dorak, 2006). Daniell et al. (2012) have demonstrated that an artificial spike to samples allows both a significant improvement in the estimation of DNA quantity and a decrease in the variability in the estimation. The aim of the current study was to develop a quantitative PCR (qPCR)-based assay for determining the biomass and number of F. candida. We conducted two experiments, one to assess the relationship between DNA quantity and collembolan number, and the other, to determine the relationship between DNA quantity and body length. The genes, nuclear RNA polymerase II (Pol II) and mitochondrial cytochrome c oxidase subunit I (mtCOI) were selected for estimation. The mtCOI gene is frequently used in
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phylogenetic studies of animals; in insects, the Pol II gene is believed to be a single-copy gene (Wiegmann et al., 2009), thus giving more precise measurements of DNA copy number (Tellenbach et al., 2010). DNA fragments of an artificial sequence were spiked into the samples to serve as the one constant control for the experimental errors and losses that occur during DNA extraction. Assays were performed on samples containing different numbers of individuals at the same body length (same biomass) and samples containing the same number of individuals at a different body length (different biomass). Biomass was estimated from the body length.
2. Materials and methods 2.1. Test organisms and culture conditions Animals for testing were obtained from laboratory stock cultures of F. candida from a population in central Japan (Itoh et al., 1995) and reared on baker0 s yeast, as described by Nakamori et al. (2008). Our laboratory population represented the offspring of a single individual. Age-synchronised collembolan specimens were prepared according to the ISO (1999). Specimens were collected in 1.5-ml tubes, preserved in 100 percent ethanol, and stored at 20 1C until use.
2.2. Experimental design For DNA quantity–number experiments, different numbers of collembolans (1, 5, 10, 20, 30, 40 and 50 individuals) at 13–16 and 34–37 days of age were collected, and two replicates for each number of individuals at each age were performed; for DNA quantity–body length experiments, 10 specimens with two replicates were collected at each age (0–3, 6–9, 13–16, 20–23, 27–30 and 34–37 days of age). To clarify the relationship between collembolan biomass and an increase in DNA quantity, age-related changes in body length were used as the parameter for assessing collembolan growth.
2.3. Measurements of body length Before DNA extraction, the length of the collembolans from mouthpart to anus was measured, placing the specimens side down in ethanol under a digital microscope, VHX-1000 (Keyence corporation, Osaka, Japan). For the different numbers of samples at the same ages, measurements were performed on 20 specimens for each age, and the mean value was used; for the body length measurements at different ages, 10 individuals with two replicates were measured.
2.4. Biomass estimation The regression between F. candida body length and dry weight was calculated assuming a power function, W ¼6.457L2.990, where L is the linear length (mm) and W is the weight (mg) (Caballero et al., 2004; Tanaka, 1970).
2.5. Sequencing The sequences of the Pol II and mtCOI genes were determined for designing PCR primers. Our population consisted of the offspring of a single individual (established in 2006). Genomic DNA was extracted from eggs using a QIAGEN DNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer0 s instructions. The parent specimen was preserved in the Tottori Prefectural Museum, Japan (No. TRPM-AAr0000630). The DNA sequence comprising a 658-bp region of the mtCOI gene was obtained following Protocol 1 reported by Nakamori (2013). For the Pol II gene, the 588-bp DNA sequence was determined in the same manner but using different primer pairs and PCR conditions. The primer pair used was Fc-PoIIGF (50 -GTCCGTCATGGTTCACTACG-30 ) and Fc-PoII-GR (50 -CTGCTGGAACCTCGTCTCTA-30 ). The reaction condition included an initial denaturation step at 95 1C for 10 min followed by 35 cycles at 95 1C for 15 s and 60 1C for 1 min with a final annealing/extension step at 60 1C for 7 min. All sequences were verified as originating from collembolans using the BLASTn algorithm of the basic local alignment search tool against GenBank. The sequences were submitted to GenBank (Accession Nos. AB845557 for the mtCOI gene; AB845285 for the Pol II gene).
2.6. Spike-in-control-preparation The PCR products from an artificial DNA sequence were used as a spike-incontrol. One artificial sequence was designed (Table 1). The DNA fragment was synthesised by GenScript USA Inc. The 83-bp DNA fragment was amplified using the ArtL2 (50 -CTTGAAGCGACCGTTACACA-30 ) and ArtR2 (50 -CGTAATTGGCCTCAGATCGT-30 ) primers. Amplification was performed in 25-ml reaction volumes containing 1 ml of DNA template, 12.5 ml of AmpliTaq Golden MasterMix (Applied Biosystems), and 25 mM of each primer. The reaction condition included an initial denaturation step at 95 1C for 10 min followed by 35 cycles at 95 1C for 15 s and 60 1C for 1 min with a final annealing/extension step at 60 1C for 7 min. The PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN), eluted with pure water and stored at 20 1C. The amplicons from the artificial sequence (20 ml, 5 10 4 ng/ml) were spiked in the test samples prior to DNA extraction. The PCR amplicons were also used for generating calibration curves in real-time PCR. The artificial sequence cannot be found in the sequences of the test samples.
2.7. DNA extraction Ethanol was removed from the screw cap microtubes (2-ml) by pipette, and the residual volume was air-dried for 10 min. Collembolan DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN) with some modifications. Sterile glass beads (0.1 mm in diameter, 0.2 g and 2.50–3.50 mm in diameter, number 8) were added to the tubes containing the samples; ATL buffer (270 ml) was added, and the contents were mixed well and placed on a shaker for 10 min at 30 Hz (1/s) frequency using a QIAGEN Tissue Lyser II. Samples were frozen for 20 min at 70 1C and thawed at room temperature, and then, the process was repeated. Thirty microlitres of proteinase K was added, and the contents were mixed well by vortexing and incubated at 56 1C overnight with rotation at 100 rpm/min in a hybrid oven. Further extraction procedures used 200 ml of mixture and followed the manufacturer0 s instructions (QIAGEN). The purified DNA was eluted into 80 ml of pure water and stored at 20 1C until use.
2.8. qPCR qPCR experiments were performed in 0.2-ml 96 well plates using the StepOnePlus Real-time PCR system (Applied Biosystems). Each reaction contained 0.95 ml of DNA as the template, 3.8 ml of water, 0.625 ml of each 100 mM forward and reverse primer (CF1, 50 -AACAACGCCACGCTTCTATT-30 and CR1, 50 - GTCGACAGGCGAAATTCTTC-30 for Pol II gene; FcCOI-A-F 50 -GGGTATGACTTGGGATCGAA-30 and FcCOI-A-R 50 -CTCCAGCAAGAACTGGAAGG-30 for mtCOI gene; ArtL2 and ArtR2 for spike-in-control), 6.25 ml of SYBR master mix and 0.25 ml of 50ROX (TOYOBO THUNDERBIRD SYBR qPCR Mix) in a final volume of 12.5 ml. The cycling parameters were as follows: an initial denaturation step at 95 1C for 10 min followed by 40 cycles at 95 1C for 15 s and 60 1C for 1 min, with a final annealing/extension step at 60 1C for 10 min. DNA melting curve analysis was performed. The primers were designed using Primer3 software (Rozen and Skaletsky, 2000). Standard curves were constructed in every run to calculate the concentrations of unknown relative to standard samples. The PCR products obtained using the qPCR primers were used as the standard samples. The DNA from the eggs of F. candida was extracted using the DNeasy Blood & Tissue kit (QIAGEN) and amplified using the AmpliTaq Gold Master Mix (Applied Biosystems), according to the manufacturer0 s instructions. The PCR conditions were the same as those used in the preparation of the spike-in-control, as described above. Standard curves were obtained in a duplicate series of six 10-fold dilutions of standard samples. Each run contained unknowns and standards. The PCR reactions were performed in technical duplicate for unknowns, and mean values were used. The data were normalised to the DNA quantity in the spike-in-control. Normalised values of all the samples were expressed relative to the mean normalised values of a reference treatment. For DNA quantity–number experiments, treatment with a single 13–16-day-old collembolan was used as the reference treatment; for DNA quantity–body length experiments, treatment with a 0–3-day-old collembolan was used as the reference treatment.
2.9. Data analysis Data were analysed using the R software (R Development Core Team, 2011). The relationships between DNA quantity–number and DNA quantity–dry weight were analysed using the linear regression formula, y¼ ax, where a is the constant, x is the number of individuals or dry weight (mg), and y is the DNA quantity. Curves for the DNA quantity–body length relationship were fitted to the data using the non-linear regression module of the R software. The formula was as follows: y¼ ax3, where a is the constant, x is the body length (mm) and y is the relative DNA quantity. The relationship of the relative ratio of mtCOI/Pol II–age was analysed based on the average data.
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3. Results
4. Discussion
3.1. DNA quantity–number
We developed a qPCR-based assay for determining the biomass of the collembolan, F. candida. We believe that this technique could be used to detect and quantify soil animals and would thus improve ecotoxicological testing. Although the qPCR-based assay represents a rapid and accurate way of estimating biomass, attention should be paid to the target genes and samples that could possibly influence the accuracy of the estimation. The biomass estimation was possible using both nuclear and mitochondrial genes, Pol II and mtCOI. Pol II gene was expected to be a good indicator of biomass because the number of nuclear genes per cell cannot change through a lifetime in animals. On the other hand, the results of the mtCOI gene assay should be interpreted with
Significantly positive linear correlations were observed between relative DNA quantity and collembolan number for the Pol II and mtCOI genes (Fig. 1; linear model, Po0.001). 3.2. DNA quantity–body length The attained cubic regression equations of relative DNA quantity to collembolan body length (mm) were y¼ 3.9024x3 (Pol II gene) and y ¼7.5167x3 (mtCOI gene; non-linear regression analysis: all the parameters of the non-linear model were significant at P o0.001; Fig. 2). 3.3. DNA quantity–biomass Significantly positive linear correlations were observed between relative DNA quantity and the dry weight of collembolan samples in each gene assay (Pol II and mtCOI; Figs. 3 and 4; linear model, Po0.001). The equations of the regression lines at different ages were slightly different (Fig. 3); using relative DNA quantity as the variable (y) and collembolan biomass (mg) as the predictor variable (x); the values were y¼0.06x (Pol II gene) and y¼0.115x (mtCOI gene). In the experiments with different numbers of individuals, 13–16-day-old and 34–37-day-old collembolans showed different slopes of regression lines (Fig. 4; linear model, P o0.001). 3.4. mtCOI/pol II The relative ratio of the mtCOI/Pol II genes changed with age (Fig. 5). The ratio was increased with age, especially at 27–30 days of age.
Fig. 2. The relationships between relative DNA quantity and collembolan body length, as measured by mitochondrial cytochrome c oxidase subunit I (mtCOI) and nuclear RNA polymerase II (Pol II) gene assays. Solid dots with a solid line represent the Pol II gene assay; hollow dots with a dashed line indicate the mtCOI gene assay; y¼3.9024x3 (Pol II gene) and y¼7.5167x3 (mtCOI gene). Each dot contains DNA from 10 collembolan individuals.
Fig. 1. The relationships between relative DNA quantity and collembolan number, as measured by mitochondrial cytochrome c oxidase subunit I (mtCOI) and nuclear RNA polymerase II (Pol II) gene assays. Solid dots with a solid line indicate 13–16-day-old collembolans; hollow dots with a dashed line represent 34–37-day-old collembolans. y¼ 1.752x (Pol II gene, 13–16 days old; R2 ¼0.9866); y¼ 7.753x (Pol II gene, 34–37 days old; R2 ¼ 0.9820); y¼2.315x (mtCOI gene at 13–16 days old; R2 ¼0.9883); y¼ 23.417x (mtCOI gene, 34–37 days of age; R2 ¼ 0.9757); Po 0.001 for all.
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caution, as mitochondrial number per cell and/or the number of mitochondrial genome per mitochondrion can change with physiological status; thus, the use of a mitochondrial gene may result in an incorrect estimation of biomass. It is known that increased mitochondrial number and mass result from the growth and division of preexisting organelles (Seo et al., 2010); mitochondrial DNA copy number is closely tied to reproduction (Lemire, 2005). In fact, the ratio of the mtCOI and Pol II genes had some variance with the growth of collembolans, but this variance was negligible for the biomass estimation in the present study. Although cautious data interpretation is needed, the mtCOI gene assays will be increasingly applicable to species identification and abundance estimation for other species.
Fig. 3. Correlations between collembolan dry weight and relative DNA quantity (same number of individuals; different ages). The regression lines were based on the calculation of body length to dry weight. Body lengths were measured at ages ranging from 0–3 to 34–37 days old. Solid dots with a solid line represent the nuclear RNA polymerase II (Pol II) gene assay; hollow dots with a dotted line indicate the mitochondrial cytochrome c oxidase subunit I (mtCOI) gene assay; y¼ 0.06x (Pol II gene) and y ¼0.115x (mtCOI gene). Each dot contains DNA from 10 collembolan individuals.
Because the mtCOI gene has been proposed as the standard barcode for animals, more and more sequence data for the gene are becoming available in public databases (Valentini et al., 2009). Egg production in the collembolan samples might cause variation in DNA quantity at 27–30 days of age. F. candida is a parthenogenetic species, and the animals become sexually mature at 21 and 24 days of age at 20 1C (Fountain and Hopkin, 2005). Around these ages, the samples before and after egg production would have different DNA quantities. Similar phenomena may occur in sexual species. Therefore, for the application of DNAbased biomass quantification methods to sexual species, further studies are required. We hypothesised that there would be differences in the DNA quantity and corresponding dry weight relationships between collembolan juvenile (13–16-day-old) and adult (34–37-day-old)
Fig. 5. The ratio of the DNA quantity from the mitochondrial cytochrome c oxidase subunit I (mtCOI) relative to the RNA polymerase II (Pol II) gene assay. Each dot contains DNA from 10 collembolan individuals.
Fig. 4. Correlations between collembolan dry weight (individual numbers: 0–50) and relative DNA quantity (different number of individuals; same age), as measured by mitochondrial cytochrome c oxidase subunit I (mtCOI) and nuclear RNA polymerase II (Pol II) gene assays. Relative DNA quantity was regressed against collembolan dry weight for each gene. Solid dots with a solid line indicate 13–16-day-old collembolans; hollow dots with a dashed line represent 34–37-day-old collembolans. y¼ 0.423x (Pol II gene, 13–16 days old; R2 ¼ 0.9705); y¼0.100x (Pol II gene, 34–37 days old; R2 ¼0.9444); y ¼0.563x (mtCOI gene, 13–16 days old; R2 ¼0.9775); y¼ 0.298x (mtCOI gene, 34–37 days old; R2 ¼ 0.9208); Po 0.001 for all.
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animals because of the changes in both cell size and cell mass with animal age. Studies in rats have suggested the partition of normal growth into three periods as follows: cell division alone, cell division with concomitant cell enlargement and cell enlargement alone with no further increase in the number of cells (Winick and Noble, 1965). Detection of differences in number can be applied to survival and avoidance tests (Natal-da-Luz et al., 2008), and the detection of differences in body length and estimated biomass can be applied to growth tests. Absolute quantification can also be used in these ecotoxicity tests, but this type of quantification requires calibration curves generated from known samples. Preparing a sample series of known numbers, biomass, and/or body length needs further study. The regression equations (DNA quantity vs. number, body length, and/or biomass) estimated in the present study could not be used directly if the sample preparation, DNA extraction, and/or PCR conditions differed in each occasion and laboratory. Our DNA quantification methods in combination with RNA analysis will help link the traditional endpoints with transcriptional responses in ecotoxicology tests. Transcriptional analysis has received increasing attention and became popular in ecotoxicology, but there is still a considerable gap between an understanding of biological effects and transcriptional responses (van Gestel, 2012; van Straalen and Roelofs, 2008). DNA can be easily co-extracted with RNA, allowing for the simultaneous analysis that would bridge the gap. In the future, molecular-based techniques from a genomic level, particularly qPCR, to predict the biomass of soil animals will be widely used. The application of our method could be huge benefit to soil studies; qPCR-based biomass estimations may not only serve as an endpoint but it could also aid in determining endpoints (survival, mobility, reproduction and respiration) for toxicity assessment. The design of species-specific primers will allow this technique to be applied to mixed collembolan species cultures in the future.
5. Conclusion In conclusion, biomass estimation was possible with both the nuclear Pol II and mtCOI gene assays. Although the quantity of the mtCOI gene was variable, it can still be used for the biomass estimation of soil animals. We believe that this technique would, thus, improve ecotoxicological testing.
Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research (No. 24241014; Project leader: Dr. M. Kamo) from the Ministry of Education, Culture, Sports, Science and Technology, Japan; and a grant from Yokohama National University and the Sasakawa Scientific Research Grant from The Japan Science Society. We are grateful to Dr. K. Ichisawa for archiving our specimen in the Tottori Prefectural Museum of Japan. The authors would like to thank T. Otaki at Yokohama National University for the support of DNA sequencing.
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