- Research article
- Open Access
Influence of temperature on development, reproduction and regeneration in the flatworm model organism, Macrostomum lignano
© The Author(s). 2019
- Received: 20 November 2018
- Accepted: 5 February 2019
- Published: 12 February 2019
The free-living marine flatworm Macrostomum lignano is a powerful model organism for use in studying mechanisms of regeneration and stem cell regulation due to its combination of biological and experimental properties, including the availability of transgenesis methods, which is unique among flatworm models. However, due to its relatively recent introduction in research, many aspects of this animal’s biology remain unknown. One such question is the influence of culture temperature on Macrostomum biology.
We systematically investigated how different culture temperatures affect development time, reproduction rate, regeneration, heat shock response, and gene knockdown efficiency by RNA interference (RNAi) in M. lignano. We used marker transgenic lines to accurately measure the regeneration endpoint, and to establish the stress response threshold for temperature shock. We found that compared to the culture temperature of 20 °C commonly used for M. lignano, temperatures of 25 °C–30 °C substantially increase the speed of development and regeneration, lead to faster manifestation of RNAi phenotypes, and increase reproduction rate without detectable negative consequences for the animal, while temperatures above 30 °C elicit a heat shock response.
We show that altering temperature conditions can be used to reduce the time required to establish M. lignano cultures, perform RNAi experiments, store important lines, and optimize microinjection procedures for transgenesis. These findings will help to optimize the design of experiments in M. lignano, and thus facilitate future research using this model organism.
- Heat shock response
- Macrostomum lignano
Flatworms (Platyhelminthes) are a large phylum in the animal kingdom (Metazoa), many of which exhibit the capacity to regenerate lost tissues and body parts . This regenerative ability has long attracted the interests of scientists, and the free-living planarian flatworms (Tricladida) Schmidtea mediterranea and Dugesia japonica in particular have been studied extensively and yielded numerous insights into the mechanisms underlying regeneration [2–5]. More recently, a non-planarian flatworm model Macrostomum lignano (Macrostopmorpha) has been introduced into the regeneration research arena, offering an attractive combination of experimental and biological features [6, 7]. Macrostomum lignano is a free-living marine flatworm capable of regeneration anterior to the brain and posterior to the pharynx . Similar to other flatworms, regeneration in M. lignano is made possible by stem cells called neoblasts . It is a small and transparent animal that is easy to culture in laboratory conditions. These features, together with the recently reported genome and transcriptome assemblies [10, 11] and the development of a robust transgenesis method  make M. lignano a versatile model organism for research on stem cells and regeneration .
Macrostomum lignano is a non-self-fertilizing hermaphrodite with a short generation time of 2–3 weeks . When cultured in standard laboratory conditions, animals lay approximately one egg per day. Embryonic development takes five days, and hatchlings reach adulthood in about two weeks. The laid eggs are fertilized, relatively large (100 μm) and follow the archoophoran mode of development ; i.e., they have a large, yolk-rich oocyte instead of separate yolk cells that supply a small oocyte. These properties of the eggs make them a good target for delivery of external agents, such as DNA, RNA and protein, by means of microinjection.
The possibility to introduce foreign genetic material and modify the genome of an animal is a highly sought-after experimental property, and an integral part of the genomic toolkit in model organisms commonly used for genetic research, such as the nematode Caenorhabditis elegans, fruit fly Drosophila melanogaster, yeast, and mouse, as it broadens the range of usable experimental approaches and greatly improves the chances of deciphering biological phenomena of interest. We recently demonstrated that microinjection of DNA into single-cell stage embryos can be used to generate transgenic M. lignano animals . The technique is efficient and robust, and stable transgenic lines can be obtained within three-to-four months, including F1 and F2 crosses. However, from the experimental perspective, it would be advantageous if the time required to generate transgenic animals could be shortened. Manipulation of culture temperature conditions is one way to approach this challenge.
Temperature is one of the most important factors influencing most biological processes. The overall range of temperatures that support active life on Earth stretches from as low as − 1.8 °C in polar regions to around 113 °C for thermophilic archaea at the other extreme [14, 15]. Most animals have specific temperature ranges that are optimal to their growth; this is because even small alterations in the temperature can lead to significant changes in animal metabolism. If these changes are sustainable for the organism, the increase in the temperature usually leads to an increase in the speed of physiological processes. The most common way of showing this relationship is using the temperature coefficient Q10 = (Rate 2 / Rate 1)10/(Temperature 2 − Temperature 1), which compares the rates of a process at a given temperature with the rate at temperature increased by 10 °C . The most popular way of using the Q10 index is by measuring oxygen consumption , but it may also be applied to various different measurements, such as electric organ discharge  or locomotor performance .
All living organisms respond to changes in environmental temperature. Perturbations in temperature will usually trigger the heat shock response pathway, which is an ancient, universal mechanism based on specialized chaperone molecules called heat shock proteins, or Hsps . These proteins can help other proteins to fold correctly, repair damaged proteins, or degrade them. They can also be a good indicator of stress that an organism undergoes .
Here we present how temperature affects development, growth, fertility, and regeneration capabilities of Macrostomum lignano. We tested the stress response to elevated temperatures by measuring the activity of the Heat shock 20 (Hsp20) gene using qRT-PCR and Hsp20::mScarlet transgene expression. Furthermore, we measured the hatching speed of M. lignano eggs when incubated at different temperatures, as well as the number of offspring produced in these conditions. We also investigated how changes in temperature influence regeneration speed in M. lignano, and the speed of the development of phenotypes upon gene knockdown by RNA interference (RNAi). Our findings establish optimal conditions for M. lignano cultures and will help inform future research using this model organism, particularly for creating transgenic animals and performing RNAi experiments.
The DV1 inbred M. lignano line used in this study has been described previously [10, 22, 23]. The NL10 and NL22 lines were previously established in our laboratory . Animals were cultured under laboratory conditions in plastic Petri dishes (Greiner), filled with nutrient-enriched artificial seawater (Guillard’s f/2 medium). Worms were fed ad libitum on the unicellular diatom Nitzschia curvilineata (Heterokontophyta, Bacillariophyceae) (SAG, Göttingen, Germany). Conditions in the climate chambers were set at 20 °C, 25 °C, 30 °C and 35 °C with constant aeration, and a 14 h/10 h day/night cycle.
The heat shock sensor construct KU#49 was created using a previously described double-promoter vector approach . The promoter region of M. lignano hsp20 homolog gene (Mlig005128.g2) was cloned using primers 5′-GGATGGATCCTCATTTATAAGCGTACCGTACT-3′ and 5′-TTATAAGCTTCATGCTGTTGTTGACTGGCGTA-3′ to drive expression of mScarlet-I red fluorescent protein, while elongation factor 1 alpha (EFA) promoter driving expression of NeonGreen fluorescent protein was used for the selection of transgenic animals. Two hundred thirty-five single-cell eggs were injected with the KU#49 plasmid as previously described , but without radiation treatment. Hatchlings were selected based on the presence of green fluorescence, and a stable transgenic line NL28 was established.
Twenty single-cell stage eggs per temperature condition were picked and transferred to single wells in a 6-well plate. They were monitored daily, and hatched worms were immediately removed from the test well. Each temperature condition was tested independently in triplicate.
To measure expression levels of the Mlig-hsp20 gene by qRT-PCR, 50 worms of the same age were selected for each of the three replicates. Animals were incubated for two hours at different temperatures (20 °C, 25 °C, 33 °C, 34 °C and 35 °C) and two more hours at 20 °C, before RNA extraction (RNeasy, Qiagen). Quantitative RT-PCR was done using the Light Cycler 480 (Roche) with 5′-CGAAGATGTCACTGAGGTCAAG-3′ and 5′-GCGCCTGCAGTAGAAGAAT-3′ as primers and GAPDH, COX and EIF as reference target genes, as previously described . Analysis of the results was performed using the qBase+ software (Biogazelle).
To monitor heat shock response using Hsp20::mScarlet transgene, NL28 transgenic animals were incubated for 2 h at different temperatures (20 °C, 25 °C to 35 °C with 1 °C interval, and 37 °C), followed by 22 h at 20 °C, and then imaged using Zeiss Axio Zoom V16 microscope with an HRm digital camera and Zeiss filter sets 38HE (FITC) and 43HE (DsRed). Images were analyzed using ImageJ software. Images were converted to 8-bits, the area of the worm was selected, and the median value of the signal was measured and visualized using box plot. Graphs were generated using the ggplot2 package for R.
Three sets of 20 freshly hatched worms were selected per line and temperature condition. They were kept in the selected temperature until the end of the experiment, a total duration of five weeks. Twice per week, the worms were transferred to new plates with fresh food, and the old plates, where animals laid eggs in the preceding time interval, were incubated until all eggs were hatched, after which the hatchlings were counted. The approach of counting hatchlings as a proxy to the number of laid eggs rather than the direct counting of eggs was chosen because it is easier and more reliable to count hatchlings than to count egg clumps.
Twelve NL22 worms expressing GFP marker in testes  were used per condition and cut above the testes. The worms were then placed in 12-well plates with fresh diatom and monitored daily. The days of the first appearance of GFP signal in the testes and in the seminal vesicle were noted and used to measure the time required for regeneration.
For the knockdown of Mlig-ddx39 gene, 15 worms per temperature condition (20 °C, 25 °C and 30 °C) were selected and treated with Mlig-ddx39 dsRNA fragments as previously described . The morphology and viability of the worms was monitored on a daily basis and any abnormalities were noted. GFP dsRNA was used as a negative control. For measuring the efficiency of Mlig-ddx-39 knockdown, qRT-PCR was performed in the same way as described above for the Mlig-hsp20 gene but with Mlig-ddx39 primers 5′-ACCCAGAGCTGCTGGACTAT-3′ and 5′-GTAGGAGCCCTTGTGACCTG-3′. For the knockdown of Mlig-sperm1 gene, twenty worms per temperature condition were treated with Mlig-sperm1 dsRNA fragments as previously described , and the number of animals with the enlarged testes were counted on day 4 of the treatment.
Establishing the temperature range
Heat shock response
Speed of embryonic development
The reproduction rate is a very important factor for a model organism, since animals with shorter generation time enable faster generation of data in genetic experiments. In addition, if animals produce large numbers of offspring, the generated data will, in most cases, have higher statistical power.
Effect of temperature on the speed of regeneration
Regeneration time, daysa
As expected, the speed of regeneration increased with temperature (Table 1). If we take the regeneration time at 20 °C as the standard rate, then for the regeneration of testes the calculated temperature coefficients are Q10 = 4 at 25 °C and Q10 = 3 for 30 °C and 35 °C. This shows that the largest effect is obtained when increasing the temperature to 25 °C, and the fastest regeneration takes place at 30 °C, without further benefit of increased temperature on the regeneration time. Hence, temperatures of 25 °C and 30 °C ought to be considered in regeneration experiments on M. lignano, as it shortens the duration of an experiment by two to three times (Table 1).
Effect of temperature on the development of RNAi phenotypes
Incubation time with dsRNA against Mlig-ddx39 gene until lethalitya, days, mean ± standard deviation
Percentage of animals with enlarged testesb after 4 days of incubation with dsRNA against Mlig-sperm1 gene
17.8 ± 1.3
16.6 ± 1.9
10.1 ± 0.6
9.5 ± 0.5
7.9 ± 0.2
7.2 ± 0.4
Temperature is a key factor in the husbandry of laboratory animals, and knowing the optimal values helps to provide them with the most suitable conditions. Changing the temperature has long been used as a method to influence the growth of model organisms, as best seen in the case of C. elegans [28, 29], and recently the role of temperature in the biology of planarian flatworm Schmidtea mediterranea was investigated .
Effects of temperature on biology of M. lignano, summary
Reproduction rate (number of hatchlings)
Heat shock response
> 20 fold
Mlig-ddx39 RNAi, days until lethality
Mlig-sperm1 RNAi, percentage of animals with enlarge testes at day 4.
Standard M. lignano cultures are kept at 20 °C and are transferred to fresh diatom every week, or twice a week when cultures are used for egg production for microinjections . In this way, a new population of worms can be expanded every two weeks if the starting culture is sufficiently dense. The generation time of two weeks can be quite limiting when experiments require large numbers of specimens, or when the starting culture has a small number of animals. This is often the case for establishing a new transgenic line, performing in situ hybridization, RNAi experiment or isolating nucleic acids and proteins. Increasing overall egg production by simply putting the cultures at higher temperature can be used as an easy method to quickly generate a large number of worms. However, one must be cautious to avoid undesirable stress response that could potentially lead to distorted results. While keeping the animals at 30 °C can result in a significant increase in the egg production, the changes in the morphology, visible already after three months, indicate that these conditions may cause too much stress to the worms. Indeed, we observed mild heat shock response activation at 30 °C, which quickly increases with further rising of incubation temperature.
Regeneration is one of the most prominent features of flatworms, and M. lignano provides a powerful experimental platform to study this phenomenon. We demonstrate that speed of regeneration increases with temperature in M. lignano, which can be taken into account and used to shorten time required for an experiment when needed. Similarly, gene knockdown by RNA interference is temperature-dependent and RNAi experiments can be accelerated by increasing the temperature. Importantly, the efficiency of gene knockdowns itself is not substantially affected by temperature, but the phenotypes develop faster, presumably due to elevated cell turnover. Notably, the dependence of the manifestation of RNAi phenotypes on cell proliferation has been previously reported in planarian flatworms .
In this study, we show that simple temperature control can significantly benefit a wide range of experiments using the flatworm model organism Macrostomum lignano. Based on our results and experience, we propose the following: (1) use 4 °C for storing the eggs and keeping them prior to microinjections; (2) use 20 °C for standard cultures that do not need rapid expansion, thus reducing the frequency of transferring the animals to a fresh food source; (3) use 25 °C for cultures that need to be quickly expanded and for the eggs that need to be hatched faster, for example microinjected eggs; (4) use 25 °C or 30 °C for RNAi experiments to observe the desired phenotype faster.
We thank Stijn Mouton for valuable comments on the manuscript. The DV1 line used in this study is a gift from Schärer laboratory.
This work was supported by the European Research Council Starting Grant (MacModel, grant no. 310765) to EB. KU was supported by the project 0324–2019-0040 from the Russian State Budget. The work on heat shock response was partially supported by the Russian Foundation for Basic Research (RFBR, grant no. 18–34-00288).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
JW, KU, LG and EB designed the study. JW, KU and LG performed experiments. JW, LG and EB analyzed the results. JW and EB wrote the manuscripts. All authors read and approved the final manuscripts.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Egger B, Gschwentner R, Rieger R. Free-living flatworms under the knife: past and present. Dev Genes Evol. 2007;217:89–104.View ArticleGoogle Scholar
- Umesono Y, Tasaki J, Nishimura K, Inoue T, Agata K. Regeneration in an evolutionarily primitive brain - the planarian Dugesia japonica model. Eur J Neurosci. 2011;34:863–9.View ArticleGoogle Scholar
- Rink JC. Stem cell systems and regeneration in planaria. Dev Genes Evol. 2013;223:67–84.View ArticleGoogle Scholar
- Dattani A, Sridhar D, Aziz AA. Planarian flatworms as a new model system for understanding the epigenetic regulation of stem cell pluripotency and differentiation. Semin Cell Dev Biol. 2018;S1084–9521(17):30441–X.Google Scholar
- Cebrià F, Adell T, Saló E. Rebuilding a planarian: from early signaling to final shape. Int J Dev Biol. 2018;62:537–50.View ArticleGoogle Scholar
- Ladurner P, Schärer L, Salvenmoser W, Rieger RM. A new model organism among the lower Bilateria and the use of digital microscopy in taxonomy of meiobenthic Platyhelminthes: Macrostomum lignano, n. Sp. (Rhabditophora, Macrostomorpha). J Zool Syst Evol Res. 2005;43:114–26.View ArticleGoogle Scholar
- Mouton S, Wudarski J, Grudniewska M, Berezikov E. The regenerative flatworm Macrostomum lignano, a model organism with high experimental potential. Int J Dev Biol. 2018;62:551–8.View ArticleGoogle Scholar
- Egger B, Ladurner P, Nimeth K, Gschwentner R, Rieger R. The regeneration capacity of the flatworm Macrostomum lignano - on repeated regeneration, rejuvenation, and the minimal size needed for regeneration. Dev Genes Evol. 2006;216:565–77.View ArticleGoogle Scholar
- Nimeth KT, Egger B, Rieger R, Salvenmoser W, Peter R, Gschwentner R. Regeneration in Macrostomum lignano (Platyhelminthes): cellular dynamics in the neoblast stem cell system. Cell Tissue Res. 2007;327:637–46.View ArticleGoogle Scholar
- Wasik K, Gurtowski J, Zhou X, Ramos OM, Delás MJ, Battistoni G, et al. Genome and transcriptome of the regeneration-competent flatworm. Macrostomum lignano Proc Natl Acad Sci. 2015;112:12462–7.View ArticleGoogle Scholar
- Grudniewska M, Mouton S, Simanov D, Beltman F, Grelling M, De Mulder K, et al. Transcriptional signatures of somatic neoblasts and germline cells in Macrostomum lignano. Elife [Internet]. 2016;5:e20607.Google Scholar
- Wudarski J, Simanov D, Ustyantsev K, de Mulder K, Grelling M, Grudniewska M, et al. Efficient transgenesis and annotated genome sequence of the regenerative flatworm model Macrostomum lignano. Nat Commun. 2017;8:2120.View ArticleGoogle Scholar
- Morris J, Nallur R, Ladurner P, Egger B, Rieger R, Hartenstein V. The embryonic development of the flatworm Macrostomum sp. Dev Genes Evol. 2004;214:220–39.View ArticleGoogle Scholar
- Schmidt-Nielsen K. Animal physiology: adaptation and environment: Cambridge University Press; 1997.Google Scholar
- Stetter KO. Hyperthermophiles in the history of life. Philos Trans R Soc B Biol Sci. 2006;361:1837–43.View ArticleGoogle Scholar
- Cossins A. Temperature biology of animals. Springer Science & Business Media; 2012.Google Scholar
- Nespolo RF. Lardies M a, Bozinovic F. Intrapopulational variation in the standard metabolic rate of insects: repeatability, thermal dependence and sensitivity (Q10) of oxygen consumption in a cricket. J Exp Biol. 2003;206:4309–15.View ArticleGoogle Scholar
- Dunlap KD, Smith GT, Yekta A. Temperature dependence of Electrocommunication signals and their underlying neural rhythms in the weakly electric fish. Apteronotus leptorhynchus. 2000;2000:152–62.Google Scholar
- Navas CA, James RS, Wakeling JM, Kemp KM, Johnston IA. An integrative study of the temperature dependence of whole animal and muscle performance during jumping and swimming in the frog Rana temporaria. J Comp Physiol B. 1999;169:588–96.View ArticleGoogle Scholar
- Richter K, Haslbeck M, Buchner J. The heat shock response: life on the verge of death. Mol Cell. 2010;40:253–66.View ArticleGoogle Scholar
- Jolly C. Role of the heat shock response and molecular chaperones in oncogenesis and cell death. J Natl Cancer Inst. 2000;92:1564–72.View ArticleGoogle Scholar
- Janicke T, Marie-Orleach L, De Mulder K, Berezikov E, Ladurner P, Vizoso DBBDB, et al. Sex allocation adjustment to mating group size in a simultaneous hermaphrodite. Evolution. 2013;67:3233–42.View ArticleGoogle Scholar
- Zadesenets KS, Vizoso DB, Schlatter A, Konopatskaia ID, Berezikov E, Schärer L, et al. Evidence for karyotype polymorphism in the free-living flatworm, macrostomum lignano, a model organism for evolutionary and developmental biology. PLoS One. 2016;11:e0164915.View ArticleGoogle Scholar
- Mouton S, Grudniewska M, Glazenburg L, Guryev V, Berezikov E. Resilience to aging in the regeneration-capable flatworm Macrostomum lignano. Aging Cell. 2018:e12739.Google Scholar
- Grudniewska M, Mouton S, Grelling M, Wolters AHG, Kuipers J, Giepmans BNG, et al. A novel flatworm-specific gene implicated in reproduction in Macrostomum lignano. Sci Rep. 2018;8:3192.View ArticleGoogle Scholar
- Rieger R, Gehlen M, Haszprunar G, Holmlund M, Legniti A, Salvenmoser W, et al. Laboratory cultures of marine Macrostomida (Turbellaria). Fortschr Zool. 1988;36.Google Scholar
- De Mulder K, Pfister D, Kuales G, Egger B, Salvenmoser W, Willems M, et al. Stem cells are differentially regulated during development, regeneration and homeostasis in flatworms. Dev Biol. 2009;334:198–212.View ArticleGoogle Scholar
- Hu PJ. Dauer. WromBook. 2007;1–19.Google Scholar
- Sulston J and JH. Methods, The nematode C. elegans. Wood WB, editor. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1988.Google Scholar
- Hammoudi N, Torre C, Ghigo E, Drancourt M. Temperature affects the biology of Schmidtea mediterranea. Sci Rep. 2018;8:14934.View ArticleGoogle Scholar
- Takano T, Pulvers JN, Inoue T, Tarui H, Sakamoto H, Agata K, et al. Regeneration-dependent conditional gene knockdown (Readyknock) in planarian: demonstration of requirement for Djsnap-25 expression in the brain for negative phototactic behavior. Dev Growth Differ [Internet]. 2007;49:383–94.View ArticleGoogle Scholar