An individual’s sexual phenotype is determined during development. Understanding which pathways are activated or repressed during the developmental stages leading to a sexually mature individual, for example by studying gene expression and how its level is biased between sexes, allows us to understand the functional aspects of dimorphic phenotypes between the sexes.

Several studies have quantified the differences in transcription between the sexes in mature individuals, showing the extent of this sex-bias and which functions are affected. There is, however, less data available on what occurs during the different phases of development leading to this phenotype, especially in species with specific developmental strategies, such as hemimetabolous insects. While many well-studied insects such as the honey bee, drosophila, and butterflies, exhibit an holometabolous development ("holo" meaning "complete" in reference to their drastic metamorphosis from the juvenile to the adult stage), hemimetabolous insects have juvenile stages that look similar to the adult stage (the hemi prefix meaning "half", referring to the more tissue-specific changes during development), as seen in crickets, cockroaches, and stick insects. Learning more about what happens during development in terms of the identity of genes that are sex-biased (are they the same genes at different developmental stages? What are their function? Do they exhibit specific sequence evolution rates? Is one sex over-represented in the sex-biased genes?) and their quantity over developmental time (gradual or abrupt increase in number, if any?) would allow us to better understand the evolution of sexual dimorphism at the gene expression level and how it relates to dimorphism at the organismic level.

Djordjevic et al (2021) studied the transcriptome during development in an hemimetabolous stick insect, to improve our knowledge of this type of development, where the organismic phenotype is already mostly present in the early life stages. To do this, they quantified whole-genome gene expression levels in whole insects, using RNA-seq at three different developmental stages. One of the interesting results presented by Djordjevic and colleagues is that the increase in the number of genes that were sex-biased in expression is gradual over the three stages of development studied and it is mostly the same genes that stay sex-biased over time, reflecting the gradual change in phenotypes between hatchlings, juveniles and adults. Furthermore, male-biased genes had faster sequence divergence rates than unbiased genes and that female-biased genes.

This new information of sex-bias in gene expression in an hemimetabolous insect allowed the authors to do a comparison of sex-biased genes with what has been found in a well-studied holometabolous insect, Drosophila. The gene expression patterns showed that four times more genes were sex-biased in expression in that species than in stick insects. Furthermore, the increase in the number of sex-biased genes during development was quite abrupt and clearly distinct in the adult stage, a pattern that was not seen in stick insects. As pointed out by the authors, this pattern of a "burst" of sex-biased genes at maturity is more common than the gradual increase seen in stick insects.

With this study, we now know more about the evolution of sex-biased gene expression in an hemimetabolous insect and how it relates to their phenotypic dimorphism. Clearly, the next step will be to sample more hemimetabolous species at different life stages, to see how this pattern is widespread or not in this mode of development in insects.

References

Djordjevic J, Dumas Z, Robinson-Rechavi M, Schwander T, Parker DJ (2021) Dynamics of sex-biased gene expression during development in the stick insect Timema californicum. bioRxiv, 2021.01.23.427895, ver. 6 peer-reviewed and recommended by Peer Community in Evolutionary Biology. https://doi.org/10.1101/2021.01.23.427895

Nearly 8 million people are suffering from Chagas disease in the Americas. The etiological agent, Trypanosoma cruzi, is mainly transmitted by triatomine bugs, also known as kissing or vampire bugs, which suck blood and transmit the parasite through their feces. Among these triatomine species, Rhodnius prolixus is considered the main vector, and many studies have focused on characterizing its biology, physiology, ecology and evolution. 

Interestingly, given that Rhodnius species feed almost exclusively on blood, their diet is unbalanced, and the insects can lack nutrients and vitamins that they cannot synthetize themself, such as B-vitamins. In all insects feeding exclusively on blood, symbioses with microbes producing B-vitamins (mainly biotin, riboflavin and folate) have been widely described (see review in Duron and Gottlieb 2020) and are critical for insect development and reproduction. These co-evolved relationships between blood feeders and nutritional symbionts could now be considered to develop new control methods, by targeting the ‘Achille’s heel’ of the symbiotic association (i.e., transfer of nutrient and / or control of nutritional symbiont density). But for this, it is necessary to better characterize the relationships between triatomines and their symbionts. 

R. prolixus is known to be associated with several symbionts. The extracellular gut symbiont Rhodococcus rhodnii, which reaches high bacterial densities and is almost fixed in R. prolixus populations, appears to be a nutritional symbiont under many blood sources. This symbiont can provide B-vitamins such as biotin (B7), niacin (B3), thiamin (B1), pyridoxin (B6) or riboflavin (B2) and can play an important role in the development and the reproduction of R. prolixus (Pachebat et al. (2013) and see review in Salcedo-Porras et al. (2020)). This symbiont is orally acquired through egg smearing, ensuring the fidelity of transmission of the symbiont from mother to offspring. However, as recently highlighted by Tobias et al. (2020) and Gilliland et al. (2022), other gut microbes could also participate to the provision of B-vitamins, and R. rhodnii could additionally provide metabolites (other than B-vitamins) increasing bug fitness. In the study from Filée et al., the authors focused on Wolbachia, an intracellular, maternally inherited bacterium, known to be a nutritional symbiont in other blood-sucking insects such as bedbugs (Nikoh et al. 2014), and its potential role in vitamin provision in triatomine bugs. 

After screening 17 different triatomine species from the 3 phylogenetic groups prolixus, pallescens and pictipes, they first show that Wolbachia symbionts are widely distributed in the different Rhodnius species. Contrary to R. rhodnii that were detected in all samples, Wolbachia prevalence was patchy and rarely fixed. The authors then sequenced, assembled, and compared 13 Wolbachia genomes from the infected Rhodnius species. They showed that all Wolbachia are phylogenetically positioned in the supergroup F that contains wCle (the Wolbachia from bedbugs). In addition, 8 Wolbachia strains (out of 12) encode a biotin operon under strong purifying selection, suggesting the preservation of the biological function and the metabolic potential of Wolbachia to supplement biotin in their Rhodnius host. From the study of insect genomes, the authors also evidenced several horizontal transfers of genes from Wolbachia to Rhodnius genomes, which suggests a complex evolutionary interplay between vampire bugs and their intracellular symbiont. 

This nice piece of work thus provides valuable information to the fields of multiple partners / nutritional symbioses and Wolbachia research. Dual symbioses described in insects feeding on unbalanced diets generally highlight a certain complementarity between symbionts that ensure the whole nutritional complementation. The study presented by Filée et al. leads rather to consider the impact of multiple symbionts with different lifestyles and transmission modes in the provision of a specific nutritional benefit (here, biotin). Because of the low prevalence of Wolbachia in certain species, a “ménage à trois” scenario would rather be replaced by an “open couple”, where the host relationship with new symbiotic partners (more or less stable at the evolutionary timescale) could provide benefits in certain ecological situations. The results also support the potential for Wolbachia to evolve rapidly along a continuum between parasitism and mutualism, by acquiring operons encoding critical pathways of vitamin biosynthesis.

References

Duron O. and Gottlieb Y. (2020) Convergence of Nutritional Symbioses in Obligate Blood Feeders. Trends in Parasitology 36(10):816-825. https://doi.org/10.1016/j.pt.2020.07.007

Filée J., Agésilas-Lequeux K., Lacquehay L., Bérenger J.-M., Dupont L., Mendonça V., Aristeu da Rosa J. and Harry M. (2023) Wolbachia genomics reveals a potential for a nutrition-based symbiosis in blood-sucking Triatomine bugs. bioRxiv, 2022.09.06.506778, ver. 3 peer-reviewed and recommended by Peer Community in Evolutionary Biology. https://doi.org/10.1101/2022.09.06.506778

Gilliland C.A. et al. (2022) Using axenic and gnotobiotic insects to examine the role of different microbes on the development and reproduction of the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae). Molecular Ecology. https://doi.org/10.1111/mec.16800

Nikoh et al. (2014) Evolutionary origin of insect–Wolbachia nutritional mutualism. PNAS. 111(28):10257-10262. https://doi.org/10.1073/pnas.1409284111

Pachebat, J.A. et al. (2013). Draft genome sequence of Rhodococcus rhodnii strain LMG5362, a symbiont of Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae), the principle vector of Trypanosoma cruzi. Genome Announc. 1(3):e00329-13. https://doi.org/10.1128/genomea.00329-13

Salcedo-Porras N., et al. (2020). The role of bacterial symbionts in Triatomines: an evolutionary perspective. Microorganisms. 8:1438. https://doi.org/10.3390%2Fmicroorganisms8091438

Tobias N.J., Eberhard F.E., Guarneri A.A. (2020) Enzymatic biosynthesis of B-complex vitamins is supplied by diverse microbiota in the Rhodnius prolixus anterior midgut following Trypanosoma cruzi infection. Computational and Structural Biotechnology Journal. 3395-3401. https://doi.org/10.1016/j.csbj.2020.10.031 

Reconstructing ancestral character states is an exciting but difficult problem. The fossil record carries a great deal of information, but it is incomplete and not always easy to connect to data from modern species. Alternatively, ancestral states can be estimated by modelling trait evolution across a phylogeny, and fitting to values observed in extant species. This approach, however, is heavily dependent on the underlying assumptions, and typically results in wide confidence intervals.

An alternative approach is to gain information on ancestral character states from DNA sequence data. This can be done directly when the trait of interest is known to be determined by a single, or a small number, of major effect genes. In some of these cases it can even be possible to investigate an ancestral trait of interest by inferring and resurrecting ancestral sequences in the laboratory. Examples where this has been successfully used to address evolutionary questions range from the nocturnality of early mammals [1], to the loss of functional uricases in primates, leading to high rates of gout, obesity and hypertension in present day humans [2]. Another possibility is to rely on correlations between species traits and the genome average substitution rate/process. For instance, it is well established that the ratio of nonsynonymous to synonymous substitution rate, dN/dS, is generally higher in large than in small species of mammals, presumably due to a reduced effective population size in the former. By estimating ancestral dN/dS, one can therefore gain information on ancestral body mass (e.g. [3-4]).

The interesting paper by Wu et al. [5] further develops this second possibility of incorporating information on rate variation derived from genomic data in the estimation of ancestral traits. The authors analyse a large set of 1185 genes in 89 species of mammals, without any prior information on gene function. The substitution rate is estimated for each gene and each branch of the mammalian tree, and taken as an indicator of the selective constraint applying to a specific gene in a specific lineage – more constraint, slower evolution. Rate variation is modelled as resulting from a gene effect, a branch effect, and a gene X branch interaction effect, which captures lineage-specific peculiarities in the distribution of functional constraint across genes. The interaction term in terminal branches is regressed to observed trait values, and the relationship is used to predict ancestral traits from interaction terms in internal branches. The power and accuracy of the estimates are convincingly assessed via cross validation. Using this method, the authors were also able to use an unbiased approach to determine which genes were the main contributors to the evolution of the life-history traits they reconstructed.

The ancestors to current placental mammals are predicted to have been insectivorous - meaning that the estimated distribution of selective constraint across genes in basal branches of the tree resembles that of extant insectivorous taxa - consistent with the mainstream palaeontological hypothesis. Another interesting result is the prediction that only nocturnal lineages have passed the Cretaceous/Tertiary boundary, so that the ancestors of current orders of placentals would all have been nocturnal. This suggests that the so-called "nocturnal bottleneck hypothesis" should probably be amended. Similar reconstructions are achieved for seasonality, sociality and monogamy – with variable levels of uncertainty.

The beauty of the approach is to analyse the variance, not only the mean, of substitution rate across genes, and their methods allow for the identification of the genes contributing to trait evolution without relying on functional annotations. This paper only analyses discrete traits, but the framework can probably be extended to continuous traits as well.

References

[1] Bickelmann C, Morrow JM, Du J, Schott RK, van Hazel I, Lim S, Müller J, Chang BSW, 2015. The molecular origin and evolution of dim-light vision in mammals. Evolution 69: 2995-3003. doi: https://doi.org/10.1111/evo.12794

[2] Kratzer, JT, Lanaspa MA, Murphy MN, Cicerchi C, Graves CL, Tipton PA, Ortlund EA, Johnson RJ, Gaucher EA, 2014. Evolutionary history and metabolic insights of ancient mammalian uricases. Proceedings of the National Academy of Science, USA 111:3763-3768. doi: https://doi.org/10.1073/pnas.1320393111

[3] Lartillot N, Delsuc F. 2012. Joint reconstruction of divergence times and life-history evolution in placental mammals using a phylogenetic covariance model. Evolution 66:1773-1787. doi: https://doi.org/10.1111/j.1558-5646.2011.01558.x

[4] Romiguier J, Ranwez V, Douzery EJ, Galtier N. 2013. Genomic evidence for large, long-lived ancestors to placental mammals. Molecular Biology and Evolution 30:5-13. doi: https://doi.org/10.1093/molbev/mss211

[5] Wu J, Yonezawa T, Kishino H. 2016. Rates of Molecular Evolution Suggest Natural History of Life History Traits and a Post-K-Pg Nocturnal Bottleneck of Placentals. Current Biology 27: 3025-3033. doi: https://doi.org/10.1016/j.cub.2017.08.043

Koutsovoulos et al. [1] have generated and analysed the first population genomic dataset in root-knot nematode Meloidogyne incognita. Why is this interesting? For two major reasons. First, M. incognita has been documented to be apomictic, i.e., to lack any form of sex. This is a trait of major evolutionary importance, with implications on species adaptive potential. The study of genome evolution in asexuals is fascinating and has the potential to inform on the forces governing the evolution of sex and recombination. Even small amounts of sex, however, are sufficient to restore most of the population genetic properties of true sexuals [2]. Because rare events of sex can remain undetected in the field, to confirm asexuality in M. incognita using genomic data is an important step. The second reason why M. incognita is of interest is that this nematode is one of the most harmful pests currently living on earth. M. incognita feeds on the roots of many cultivated plants, including tomato, bean, and cotton, and has been of major agricultural importance for decades. A number of races were defined based on host specificity. These have played a key role in attempts to control the dynamic of M. incognita populations via crop rotations. Races and management strategies so far lack any genetic basis, hence the second major interest of this study.
The authors newly sequenced the full genome of eleven strains from Brazil and added nine already available samples from Africa and North-America. They report that, in all likelihood, M. incognita is indeed a purely asexual species. This is supported by (i) the confirmation that the genome is in its major part haploid, and (ii) a spectacularly high level of linkage disequilibrium, which does not decline with genetic distance between loci at a 100kb scale. The absence of sex and recombination is associated in M. incognita with a remarkably low amount of genetic diversity - one order of magnitude less than in typical sexual nematodes - and an heavy load of deleterious mutations, as measured by the ratio of non-synonymous (=amino-acid changing) to synonymous (=amino-acid conservative) diversity in coding sequences. The other important result of this study is that the population substructure in M. incognita is in no way related to host races or geography. The tree genetic clusters that are identified include strains from several continents and feeding on a diversity of host plants.
The implications of this work are numerous. First, the results suggest that M. incognita is an ancient asexual. Asexuality, which was here demonstrated via linkage disequilibrium analysis, must be ancient enough for diploidy (or, in this case, maybe triploidy) to have been lost - i.e., formerly homologous chromosomes have accumulated enough mutations to be assembled as distinct entities. So we are not talking about a highly successful clone having recently spread the world - rather a long-term obligate parthenogen. Asexual organisms are deprived of the source of genetic variation offered by recombination, which is why asexuality is thought to be an evolutionary dead-end. Long-term asexuals are uncommon and even the most famous ones, bdelloid rotifers, are suspected to experience between-individual genetic transfers [3]. M. incognita is apparently a true 'evolutionary scandal', and as such deserves particular attention from molecular evolutionary geneticists.
The lack of any host race effect on the genetic diversity of M. incognita is another important finding. So-called 'races' have largely contributed to shape researchers' view of the structure of the species so far. This study demonstrates that a mental effort is now needed to forget about races, and consider host-specificity for what it is - a phenotypic trait. This result implies that many host shifts must have independently occurred in the three M. incognita genetic lineages, suggesting an arms race between plants and nematodes, which in the absence of sex and recombination must be entirely mutation-driven on the nematode side. Genes functionally involved in the arms race might therefore be expected to have experienced convergent evolution, if distinct M. incognita lineages have adopted the same solutions to overcome plant defenses. The present study paves the way for such a genome scan. The authors rightly discuss that the strong adaptive potential of M. incognita, at least in terms of host shift, despite no sex and tiny amounts of genetic diversity, is a paradox that would deserve to be further investigated.

References

[1] Koutsovoulos, G. D., Marques, E., Arguel, M. J., Duret, L., Machado, A. C. Z., Carneiro, R. M. D. G., Kozlowski, D. K., Bailly-Bechet, M., Castagnone-Sereno, P., Albuquerque, E. V., & Danchin, E. G. J. (2019). Population genomics supports clonal reproduction and multiple gains and losses of parasitic abilities in the most devastating nematode plant pest. bioRxiv, 362129, ver. 5, peer-reviewed and recommended by Peer Community in Evolutionary Biology. doi: 10.1101/362129
[2] Hartfield, M. (2016). Evolutionary genetic consequences of facultative sex and outcrossing. Journal of evolutionary biology, 29(1), 5-22. doi: 10.1111/jeb.12770
[3] Debortoli, N., Li, X., Eyres, I., Fontaneto, D., Hespeels, B., Tang, C. Q., Flot, J. F. & Van Doninck, K. (2016). Genetic exchange among bdelloid rotifers is more likely due to horizontal gene transfer than to meiotic sex. Current Biology, 26(6), 723-732. doi: 10.1016/j.cub.2016.01.031

In partially clonal organisms, genetic markers are often used to characterize the genotypic diversity of populations and infer thereof the relative importance of clonal versus sexual reproduction. Most studies report a measure of genotypic diversity based on a ratio, R, of the number of distinct multilocus genotypes over the sample size, and qualitatively interpret high / low R as indicating the prevalence of sexual / clonal reproduction. However, a theoretical framework allowing to quantify the relative rates of clonal versus sexual reproduction from genotypic diversity is still lacking, except using temporal sampling. Moreover, R is intrinsically highly dependent on sample size and sample design, while alternative measures of genotypic diversity are more robust to sample size, like D*, which is equivalent to the Gini-Simpson diversity index applied to multilocus genotypes. Another potential indicator of reproductive strategies is the inbreeding coefficient, Fis, because population genetics theory predicts that clonal reproduction should lead to negative Fis, at least when the sexual reproduction component occurs through random mating. Taking advantage of this prediction, Arnaud-Haond et al. [1] reanalysed genetic data from 165 populations of four partially clonal seagrass species sampled in a standardized way. They found positive correlations between Fis and both R and D* within each species, reflecting variation in the relative rates of sexual versus clonal reproduction among populations. Moreover, the differences of mean genotypic diversity and Fis values among species were also consistent with their known differences in reproductive strategies. Arnaud-Haond et al. [1] also conclude that previous works based on the interpretation of R generally lead to underestimate the prevalence of clonality in seagrasses. Arnaud-Haond et al. [1] confirm experimentally that Fis merits to be interpreted more properly than usually done when inferring rates of clonal reproduction from population genetics data of species reproducing both sexually and clonally. An advantage of Fis is that it is much less affected by sample size than R, and thus should be more reliable when comparing studies differing in sample design. Hence, when the rate of clonal reproduction becomes significant, we expect Fis < 0 and D* < 1. I expect these two indicators of clonality to be complementary because they rely on different consequences of clonality on pattern of genetic variation. Nevertheless, both measures can be affected by other factors. For example, null alleles, selfing or biparental inbreeding can pull Fis upwards, potentially eliminating the signature of clonal reproduction. Similarly, D* (and other measures of genotypic diversity) can be low because the polymorphism of the genetic markers used is too limited or because sexual reproduction often occurs through selfing, eventually resulting in highly similar homozygous genotypes.
The work of Arnaud-Haond et al. [1] shows that the populations genetics of partially clonal organisms should be better studied, an endeavour encompassed in a companion paper using numerical simulations [2]. A further step that remains to be accomplished is to build a mathematical framework for developing estimators of rates of clonal versus sexual reproduction based on genotypic diversity.

References

[1] Arnaud-Haond, S., Stoeckel, S., and Bailleul, D. (2019). New insights into the population genetics of partially clonal organisms: when seagrass data meet theoretical expectations. ArXiv:1902.10240 [q-Bio], v6 peer-reviewed and recommended by Peer Community in Evolutionary Biology. Retrieved from http://arxiv.org/abs/1902.10240
[2] Stoeckel, S., Porro, B., and Arnaud-Haond, S. (2019). The discernible and hidden effects of clonality on the genotypic and genetic states of populations: improving our estimation of clonal rates. ArXiv:1902.09365 [q-Bio], v4 peer-reviewed and recommended by Peer Community in Evolutionary Biology. Retrieved from http://arxiv.org/abs/1902.09365