Guests in the aphid room…. the digital PCR

After the PCR discovery due to Kary Mullis, several improvements have been obtained in order to amplify and quantify nucleic acids. Among them, the Real-Time PCR, also called quantitative polymerase chain reaction (qPCR), is probably one of the most powerful and sensitive techniques for quantitative gene expression analysis and pathogen detection.

As the name suggests, the real-time PCR measures PCR amplification as it occurs, at the contrary of what happens on standard PCR, where results are collected after the reaction has been completed, so making impossible to determine the starting concentration of nucleic acid. On the contrary Real-Time PCR focusses on the exponential phase of the amplification and calculates the Threshold line (that is the level of detection at which a reaction reaches a fluorescent intensity above background) and the PCR cycle at which the sample reaches this level that is called the Cycle Threshold (Ct). The Ct value is used in downstream quantitation or presence/absence detection. By comparing the Ct values of samples of unknown concentration with a series of standards, the unknown amount of template DNA can be accurately determined.

It is quite easy in theory, but not so easy in practice… since the choice of controls, normalization methods and quality control management have serious implications for the reliability, relevance and reproducibility of qPCR experiments so that two researchers can perform the same real-time PCR experiment and get different results (as reported here in BioTechniques). I’m not trying to convince you that the qPCR does not work, simply I do not like this technique at least for my in vivo experiments in aphids.

This is why I’m reading with great interest the lastest news about digital PCR that could represent a relevant improvement for DNA quantitation in respect to qPCR. Digital PCR is a new approach to nucleic acid detection and quantification that differs in respect to qPCR, because it directly counts the number of target molecules rather than relying on reference standards or endogenous controls (qPCR vs digital PCR at a glance from Invitrogen). As you can see in the figure below (from Invitrogen), digital PCR works by partitioning a sample into many individual  PCR reactions; some portions of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative answers is used to generate an absolute answer for the exact number of target molecules in the sample, without reference to standards or endogenous controls.

As summarized by Monya Baker in Nature Methods (2012):

“Digital PCR (dPCR) uses the same primers and probes as qPCR but is capable of higher sensitivity and precision. In standard implementations, qPCR cannot distinguish gene expression differences or copy number variants smaller than about twofold. Identifying alleles with frequencies of less than about 1% is difficult because such tests would also detect highly abundant common alleles with similar sequences. In contrast, dPCR can measure a 30% or smaller difference in gene expression, distinguish whether a variant occurs in five versus six copies and identify alleles occurring at a frequency of one in thousands. It can also be used to standardize qPCR assays.”

The more partitions, the greater the resolution. For instance with new techniques for digital PCR your sample will be partitioned in 20.000 droplets (as reported here by Biorad), each working as a single qPCR experiment that means that you could have a quantitative analysis similar for precision of doing 20,000 replicates of a quantitative PCR!

Bio-Rad (using the QuantaLife technology) has machines that offer many sample partitions using a sort of droplet digital PCR, where reaction chambers are separated not by the walls of a well but by carefully titrated emulsions of oil, water and stabilizing chemicals. First, samples are put into a machine where they are mixed with all the necessary reagents and dispersed into tiny droplets. The droplets for each sample are transferred into tubes that can be placed in a thermocycler for PCR. Afterward, the tubes are transferred to a droplet reading machine, which functions like a flow cytometer to analyze each droplet for whether or not a reaction has occurred. Would you like to know something more? Look at this nice Biorad video:

At the moment, digital PCR is a specialist approach that is much more costly than quantitative PCR mainly for the digital PCR apparatus (you have to pay about 100.000 €) rather than for the PCR chemistry, but as the next generation sequencing recently showed, as the technology matures… the costs come down so that there are few users for digital PCR apparatus today, but there will be more tomorrow.

Baker, M. (2012). Digital PCR hits its stride Nature Methods, 9 (6), 541-544 DOI: 10.1038/nmeth.2027

A DNA sequence (even if long) isn’t enough to understand a genome

Today I was reading a paper entitled “Genomes in three dimensions” published by Monya Baker in Nature about two years ago. According to her commentary, we must study the chromosome structure to understand how a genome works, so that we must have data not only consisting of DNA sequences, but also genomes in three dimensions.

In particular, the commentary focussed on studies about three-dimensional structures of chromosomes coiled in the nucleus… since before scientists can really understand what sequencing data tell us, we have to study the chromosome interactions in the nucleus, the higher-order chromosome arrangements and the long-range looping interactions that bring gene sequences into physical contact with far-off regulatory elements.

We know, for instance, that inactive chromatin is generally located near the nuclear periphery, so that translocations of genes within chromosomal regions near the nuclear envelope may switch off them. At the same time, we know that long-range interactions can be altered by disease-associated mutations in stretches of DNA that do not code for genes.

This is true… and that is not the full story! We know that some chromosomal regions are more prone to rearrangement than others. Moreover, the evolution of genes is not necessarily the same on each chromosomes. For instance Singh (2005) showed that the comparison of the patterns of molecular evolution between autosomes and sex chromosomes (such as X and W chromosomes) can provide insight into the forces underlying genome evolution. In particular, he investigated the patterns of codon bias evolution on the X chromosome and autosomes in Drosophila melanogaster and Caenorhabditis elegans assessing that X-linked genes have significantly higher codon bias compared to autosomal genes in both flies and nematodes. Moreover, DNA sequences on X chromosomes often have a faster rate of evolution when compared to similar loci on the autosomes, as assessed by Meisel et al. (2012). Similarly, Mank et al. reported that evolution proceeds more quickly on gene located in the Z chromosome in birds (with ZZ males and ZW females), where hemizygous exposure of beneficial nondominant mutations increases the rate of fixation, as well as genes on the human X chromosomes evolved rapidly in comparison to genes located on autosomes (Kvikstad and Makova 2008). Finally, each chromosome may have differential evolution rates in diverse regions. Indeed, Casto and colleagues (2010) highlighted two X-chromosomal regions that are outliers relative to the rest of the human X chromosome with respect to the distribution of mutations suggesting that these loci were differentially influenced by selection in the past.

In the last years, several genome projects have been published without any kind of information about the localization of the annotated genes. Even if the availability of a wholly sequenced genome is a powerful tool to study the evolution of an organism, the absence of information related to the gene mapping (together with the absence of interest for gene mapping) makes genome projects less effective than someone think. What a missed opportunity!

Singh, N. (2005). X-Linked Genes Evolve Higher Codon Bias in Drosophila and Caenorhabditis Genetics, 171 (1), 145-155 DOI: 10.1534/genetics.105.043497

Meisel RP, Malone JH, & Clark AG (2012). Faster-X evolution of gene expression in Drosophila. PLoS genetics, 8 (10) PMID: 23071459

Mank, J., Axelsson, E., & Ellegren, H. (2007). Fast-X on the Z: Rapid evolution of sex-linked genes in birds. Genome Research, 17 (5), 618-624 DOI: 10.1101/gr.6031907

Baker, M. (2011). Genomics: Genomes in three dimensions Nature, 470 (7333), 289-294 DOI: 10.1038/470289a

Casto AM, Li JZ, Absher D, Myers R, Ramachandran S, & Feldman MW (2010). Characterization of X-linked SNP genotypic variation in globally distributed human populations. Genome biology, 11 (1) PMID: 20109212

Evolving aphids: one genome-one organism insects or holobionts?

Recently I published with the colleague and friend Gian Carlo Manicardi a short review in Invertebrate Survival Journal about the relevant role of symbionts in the evolution of aphids.

Aphids have obligate mutualistic relationships with microorganisms that provide them with essential substances lacking in their diet, together with symbionts conferring them conditional adaptive  advantages related, for instance, to the thermal tolerance and to the resistance to parasitoid wasps.  The presence/absence of a secondary symbiont may have a relevant phenotypic effect so that aphid  microbial symbionts constitute a sort of second genome with its own genetic inheritance.

On the whole, genes important for aphid survival and reproduction are not uniquely present in the aphid nuclear and mitochondrial genomes, but also in the chromosomes of each symbiont. As a consequence, aphids should be viewed as holobionts with an extended genome (the hologenome)  including the host and its symbiotic microbiome. In this connection, the true unit of selection in evolution must be considered the aphid holobiont, in place of the single host as individual separated from its symbionts.

Mandrioli M, Manicardi GC (2013). Evolving aphids: one genome-one organism insects or holobionts? Invertebrate Survival Journal, 10, 1-6 (free pdf available here)

Make your choice.. of plants!

Aphids harbour several obligate and facultative bacterial symbionts that have important effects on their life. Several surveys of secondary symbionts clearly show that particular species are strongly associated with aphids feeding on certain food plants. For instance, most pea aphid clones feeding on clover Trifolium sp. harbour Regiella insecticola, while those feeding on Medicago usually have Hamiltonella defensa.

How can we explain such a difference? The most intriguing hypothesis is that these patterns reflect a role of these symbionts in the host plant use. However, they may also be present in view of factors correlated with host plant use or simple historical contingency. So the question is: Can symbiont drive the choice of the plant by aphids or they simply change in view of the plants where aphids live?

Several studies tried to distinguish between these explanations furnishing controversial scenarios. Tsuchida et al. (2004) removed R. insecticola from a clover-associated pea aphid clone using antibiotics and found that performance on Trifolium, but not Vicia, was negatively affected. In the same year, Leonardo repeated the same experimental plan but without finding any fitness effects of removing R. insecticola from two clones of aphid specialized on Trifolium. With a different approach, based on the artificial introduction of R. insecticola into five symbiont-free clones not previously associated with clover, no effect on performance of aphids on Trifolium have been observed by Ferrari et al. (2007). These results, as a whole, suggested that symbionts may be involved in the plant choice but not alone. Probably, interactions between aphids and plants involve the genotype of either the host or symbiont and both can influence host plant use.

Ferrari and Godfray here reported a further set of experiments where they evaluated the fitness consequences of introducing different strains of the symbiont Hamiltonella defensa into three aphid clones (that naturally lack symbionts) collected on Lathyrus pratensis and of removing symbionts from 20 natural aphid–bacterial associations. Ferrari and Godfray reported that: “Infection decreased fitness on Lathyrus but not on Vicia faba, a plant on which most pea aphids readily feed. This may explain the unusually low prevalence of symbionts in aphids collected on Lathyrus. There was no effect of presence of symbiont on performance of the aphids on the host plants of the clones from which the H. defensa strains were isolated. Removing the symbiont from natural aphid–bacterial associations led to an average approximate 20 per cent reduction in fecundity, both on the natural host plant and on V. faba, suggesting general rather than plant-species-specific effects of the symbiont. Throughout, we find significant genetic variation among aphid clones”.

Can you have now a better scenario? As a whole, the results provide no evidence that secondary symbionts have a major direct role in facilitating aphid utilization of particular host plant species, but only the aphid genome seem to have a pivotal role in the plant choice. At present we have a reply, but further experiments on different aphid species are welcome!

References

McLean, A., van Asch, M., Ferrari, J., & Godfray, H. (2010). Effects of bacterial secondary symbionts on host plant use in pea aphids Proceedings of the Royal Society B: Biological Sciences, 278 (1706), 760-766 DOI: 10.1098/rspb.2010.1654
Tsuchida, T. (2004). Host Plant Specialization Governed by Facultative Symbiont Science, 303 (5666), 1989-1989 DOI: 10.1126/science.1094611
Leonardo, T. (2004). Removal of a specialization-associated symbiont does not affect aphid fitness Ecology Letters, 7 (6), 461-468 DOI: 10.1111/j.1461-0248.2004.00602.x
Ferrari, J., Scarborough, C., & Godfray, H. (2007). Genetic variation in the effect of a facultative symbiont on host-plant use by pea aphids Oecologia, 153 (2), 323-329 DOI: 10.1007/s00442-007-0730-2

Insects? Not just one genome-one organism

In the last day I read with great interest the intriguing review entitled “A symbiotic view of life: we have never been individuals” written by  Scott F. Gilbert, Jan Sapp and Alfred I. Tauber and published in The Quarterly Review of Biology.

Due to their parthenogenetic reproduction aphids are generally considered a sort of clone so that each individual is identical to the others in the population. According to this suggestion, several Authors refereed to aphids as a single genome species. Actually, as well stated by Gilbert et al, the one-genome/one-organism doctrine of classical genetics has been eclipsed by recent studies on symbiosis. In particular, aphid microbial symbionts form a second type of genome and genetic inheritance (Moran 2007; Gilbert 2011). As frequently suggested, insects may acquire their symbionts vertically though the maternal germline as well as horizontally from the environment (such as during feeding). In particular, in aphids, symbiotic bacteria provide selectable allelic variation (thermotolerance, color, parasitoid resistance) that enables some hosts to persist better under different environmental conditions (Dunbar et al. 2007; Tsuchida et al. 2010).

A well-studied example is the pea aphid, Acyrthosiphon pisum since variants of its symbiont Buchnera provide the aphid with thermotolerance, even if at the expense of fecundity at normal temperatures; Dunbar et al. 2007). The second bacterial symbiont Rickettsiella is responsible for color change, turning genetically red aphids into green through the synthesis of quinones (Tsuchida et al. 2010). Furthermore, variants of Hamiltonella symbionts provide immunity against parasitoid wasp infection (Oliver et al. 2009). Interestingly, in the last case, the protective role of Hamiltonella is due to the incorporation of a specific lysogenic bacteriophage within the bacterial genome. The aphids are therefore infected by Hamiltonella that must be infected by phage APSE-3. As Oliver et al. (2009) wrote, “In our system, the evolutionary interests of phages, bacterial symbionts, and aphids are all aligned against the parasitoid wasp that threatens them all. The phage is implicated in conferring protection to the aphid and thus contributes to the spread and maintenance of H. defensa in natural A. pisum populations” .

However, symbioses are frequently not for free for the hosts and even if aphids have some advantages due to symbionts in the presence of parasitoids having their beneficial protection, in the absence of parasitoid wasps aphids carrying the bacteria with lysogenic phage are not as fecund as those lacking them. Similarly, a trade-off occurs in aphids that carry the thermotolerant genetic variants of Buchnera, meaning that more heat-resistant aphids have less fecundity at milder temperatures than their sisters whose bacteria lack the functional allele for the heat-shock protein.

As Gilber et al reminded at the ned of their review, not all scientists involved in evolution agree about the important role of symbiosis so that, for instance, in the 2009 “Homage to Darwinism” debate held at Oxford University, Richard Dawkins questioned the bringing of symbiosis into evolutionary theory: “Take the standard story for ordinary animals, [where] you’ve got a distribution of animals [and] you’ve got a promontory, or  an  island or something and so you end up with two [geographical] distributions. And then on either side you get different selection pressures, and so one [group] starts to evolve this way, and [the other] one starts to evolve that way, and what’s wrong with that? It’s highly plausible, it’s economical, it’s parsimonious. Why on Earth would you want to drag in symbiogenesis when it’s so unparsimonious and uneconomical?”. As Lynn Margulis replied at that time…  simply because symbiosis exists and it is common in living organisms.

 References

• Dunbar HE, Wilson AC, Ferguson NR, & Moran NA (2007). Aphid thermal tolerance is governed by a point mutation in bacterial symbionts. PLoS biology, 5 (5) PMID: 17425405

• Gilbert S. F. 2011. Symbionts as genetic sources of hereditable variation. pp. 283–293. In Transformations of Lamarckism: from sbtle fluids to molecular biology, edited by S. B. Gissis and E. Jablonka. Cambridge (Massachusetts): MIT Press.

• Moran NA (2007). Symbiosis as an adaptive process and source of phenotypic complexity. Proceedings of the National Academy of Sciences of the United States of America, 104 Suppl 1, 8627-33 PMID: 17494762

• Oliver, K., Degnan, P., Hunter, M., & Moran, N. (2009). Bacteriophages Encode Factors Required for Protection in a Symbiotic Mutualism Science, 325 (5943), 992-994 DOI: 10.1126/science.1174463

• Tsuchida T., Koga R., Horikawa M., Tsunoda T., Maoka T., Matsumoto S., Simon J.-C., Fukatsu T. 2010. Symbiotic bacterium modifies aphid body color. Science 330:1102–1104.

Fight together or escape? an insect affair!

Fungi specialised to attack insects (in the photo from the blog Hyphal Happenings) are commonly present in the environment so that they have driven many aspects of the insect evolution, affecting behavioural, chemical and immune systems.

In a recent paper published in PLoS One, Christine Turnbull and colleagues compared the activity of cuticular antifungal compounds in thrips species (Insecta: Thysanoptera) representing a gradient of increasing group size and sociality: solitary, communal, social and eusocial, against the entomopathogen Cordyceps bassiana. Solitary and communal species showed little or no activity. In contrast, the social and eusocial species killed this fungus, suggesting that the evolution of sociality has been accompanied by sharp increases in the effectiveness of antifungal compounds. This paper suggests a new insight into the evolution of thrips sociality since traits enabling nascent colonies to defend themselves against microbial pathogens should be considered essential for social evolution. Are fungal entomopathogens an integral part in the evolution of insect sociality in general?

Interestingly, a very different response has been reported by Hatano and colleague in aphids where entomopathogenic fungi stimulate transgenerational wing induction in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae)  allowing aphids to leave patches containing entomopathogenic fungi. Indeed, pea aphids infected with pathogens and maintained in groups on broad bean plants produced a higher proportion of winged morphs than uninfected control aphids.

Wing induction in aphids has been also related to the presence of predators and parasitoids, but unlike predators and parasitoids the effect of  entomopathogenic fungi is independent of physical contact with other aphids, suggesting that physiological cues induce wing formation in infected aphids. Indeed, when maintained in isolation, aphids infected with fungi pathogens also produced higher proportions of winged offspring than control aphids.

As a whole, specialised fungal entomopathogens intensified the degree of sociality and group size in some insects enabling primitive nascent colonies to combat microbial pathogens whereas the same selective agent prompted aphids (that are generally not social insects) to induce wings also in isolated aphids in order to quickly escape infected patches.

Therefore, if you see fungi pathogens fight them with our family or escape them!

References

Turnbull C, Wilson PD, Hoggard S, Gillings M, Palmer C, Smith S, Beattie D, Hussey S, Stow A, Beattie A (2012). Primordial enemies: fungal pathogens in thrips societies. PloS one, 7 (11) PMID: 23185420
Hatano E, Baverstock J, Kunert G, Pel, J, Weisser W. (2012). Entomopathogenic fungi stimulate transgenerational wing induction in pea aphids, Acyrthosiphon pisum (Hemiptera: Aphididae) Ecological Entomology, 37 (1), 75-82 DOI: 10.1111/j.1365-2311.2011.01336.x

Social behaviour and plant morphology: a case of extended phenotype in insects?

Some days ago I received a twit from Marleen Roelofs suggesting me a paper published in Nature Communications and related to aphids. The paper, entitled “An insect-induced novel plant phenotype for sustaining social life in a closed system” is extremely intriguing and deals about the evolution of aphids living inside galls.

Gall aphids (in the photo from the Yavapai County Homepage)  represent a primitive insect social society based on the construction of a completely closed gall on a plant and from hundreds to thousands of aphids grow inside this unusual nest and reproduce for several months in isolation.

Due to their diet enriched in sugars, aphids generally need to produce honeydew that is excreted forming a sticky coating on leaves and many gall-forming social aphids have small openings in their galls through which soldier nymphs actively dispose honeydew droplets and other colony wastes. Surprisingly, some gall aphids live within completely closed galls… but why these aphids are not drowned by accumulated honeydew?  This is a good question since it could be expected that the large quantity of honeydew excreted by hundreds of aphids would quickly fill up the closed gall cavity. Furthermore, when the same aphids were placed on an artificial feeding system consisting of a liquid artificial diet sandwiched by Parafilm membranes, a number of honeydew droplets soon appeared around the insects.

Where is honeydew? Mayako Kutsukake and colleagues identified the sophisticated biological solution that aphids evolved to the waste problem in the closed gall system.

The gall-forming insects manipulate the plant growth and morphogenesis for their own sake in a sophisticated manner, thereby inducing elaborate plant structures as ‘extended phenotypes’ of the insects. During their nest construction, the gall inner surface is specialized for absorbing water, whereby honeydew is promptly removed via the plant vascular system. This plant-mediated waste removal is an efficient mechanism of nest cleaning, which can be regarded as ‘extended phenotype’ and ‘indirect social behavior’ of the social aphids , but seems also to be a sort of trade-off between plants and aphids since plants can al least recover a portion of the sugars that aphids have stolen by feeding.  By contrast, no such water absorption was observed for the open galls where honewdew can be eliminated without any problem.

Interestingly, the aphid species examined in this study represent the tribes Nipponaphidini, Hormaphidini and Cerataphidini and the analysis of the complete set of data indicated that water-absorbing closed galls evolved at least twice independently among social aphids.

I found this paper simply amazing since it shows an elegant strategy for social insect colonies to persist for a considerable period in complete isolation. This is not an easy goal since (except for the inactive hibernation period), insects constantly require a large amount of food from the environment and also produce a large amount of wastes to be disposed outside. By contrast, in gall forming aphids, the plant-made nests directly provide a constant and high-quality food supply, a physical barrier against predators and parasites, mitigated environmental stresses and a mechanism for cleaning galls, the latter enabling them to evolve a unique strategy of social living in completely closed galls.

Reference

Kutsukake, M., Meng, X., Katayama, N., Nikoh, N., Shibao, H., & Fukatsu, T. (2012). An insect-induced novel plant phenotype for sustaining social life in a closed system Nature Communications, 3, 1187-1192 DOI: 10.1038/ncomms2187