Host-microbiome-pathogen interactions in cockroaches

Traditionally, philosophy uses the discrimination of self and non-self to define individuality with the immune system performing this discrimination. In the evolutionary field of biology this distinction is not that simple. Nowadays it is becoming more and more apparent that individuals can no longer be considered as ‘lone isolated islands’ in the ‘environmental sea’. All kinds of eukaryotic taxa harbour their own microbiota consisting of bacteria, archaea, fungi, protozoa and viruses and they are tolerated by the host’s immune system because of their manifold beneficial functions on, for example, host nutrition, detoxification, development, fecundity or pathogen protection. However, not only the beneficial microbiome, but also the host’s nutrition can strongly affect its physiology and its ability to combat pathogen infections. Microbiome and host form a unit – the holobiome. Notably, even though we gained insights on either the function of the microbiome or of the nutrition on host immune defence in diverse separate studies we still poorly understand how they act together in particular organisms. An insect model system to study these interactions are cockroaches. This is because, 1) they are omnivorous generalists, which makes them easily accessible for nutritional studies; 2) they harbour a diverse microbiota which can be manipulated through sterilization methods; and 3) they feature effective strategies to combat pathogens since they are frequently exposed to a rich antigenic environment due to their lifestyle. First, in Chapter I I investigated the nutritional dependencies of immunity in the cockroach system by performing food choice experiments using the cockroach species Blatta orientalis upon exposure to the opportunistic Gram-negative bacterial insect pathogen Pseudomonas entomophila. I could show that depending on the strength of infection B. orientalis males reduce their overall nutrient intake and increase the protein to carbohydrate ratio being consumed. Interestingly, these behavioural shifts do not boost the insect’s immunity as indicated by the examination of the hemolymph’s antimicrobial activity, the abundance of immune proteins in the hemolypmph or the general host survival. This lack of benefits for the host highlights a possible decoupling of dietary macronutrient regulation from immunity in these invasive animals with the possibility that anorexia, in general, might be a more powerful tool if diet quality is highly unpredictable for generalist species. In Chapter II I evaluated two different approaches for the development of a germ-free Blatella germanica cockroach breeding system which forms the basis of any study dealing with the function of the cockroach microbiome. While one of these methods uses peracetic acid, the other one uses a combination of peracetic acid and sodium hypochlorite to surface-sterilize cockroach oothecas to deprive the hatchlings from their natural microbiota. These treatments should leave them only with their obligate symbiont Blattabacterium sp., which supplies essential vitamins and is required for the development into fecundant adults. I tested the success of those techniques by plating adult individuals on Lysogeny-broth-agar and by using state of art 16S metabarcoding. It turned out that both methods performed quite poorly leading to individuals which can be considered as germ-free in 40 % of all cases. I therefore developed our own method by combining sequential ootheca surface sterilizations with peracetic acid and sodium hypochlorite followed by a treatment of freshly hatched nymphs with the antibiotics rifampicin and gentamicin which significantly improved its effectiveness resulting in germ-free adult cockroaches to 99 % of all cases. In addition, I used our germ-free cockroach system for an early study on the impact of the absence of an intact microbiome on developmental time. I could show that B. germanica cockroaches deprived of their natural microbiota needed approximately 35 days longer from the day of hatching to the day molting into adults than their conventional counterparts, which already grants a small glimpse on the strong impacts of the microbiome on the host physiology and its overall performance. In Chapter III I analysed the transcriptome of germ-free and conventional B. germanica males and followed their survival upon P. entomophila systemic infection to gain further insights on the influence of the cockroach microbiome on host traits. The basis of our gene identification were two published genomes either the one by Harrison et al. (2018) or the one by He (2018). Depending on the reference genome used for the analyses small differences existed. When the Harrison et al. genome was used 25451 putative genes were identified and 184 of those including 19 immune-related genes were significantly different expressed between conventional and germ-free cockroaches. When the He genome was used 111778 putative genes were identified and 1082 of those including 30 immune-related genes were significantly different expressed between conventional and germ-free cockroaches. Immune-related genes which were significantly expressed between germ-free and conventional cockroaches identified with both reference genomes included hemolymph lps-binding protein related genes which were mostly upregulated in germ-free individuals because of their role in trapping the Blattabacterium sp. endosymbiont and tenecin-1 genes, a transferrin, a caspase-1, a alpha-1-macroglobulin, a lysozym c-1 as well as a catalase found to be upregulated in conventional individuals. All the latter ones contribute to the recognition and the suppression of microbial life to maintain a stable host microbiota. This regulation of gene expression by the microbiome might also assist the host in combating infections as indicated by the significantly higher survival of conventional cockroaches infected with P. entomophila. In conclusion I was able to show, that host biology is heavily shaped by microbial life. Those microbes can be either invading pathogens or commensal or beneficial microbiota. In all cases they radically alter host behaviour, development and/or physiology. While pathogens only harm their hosts, the microbiome promotes host phenotypes like development or immune competence. Therefore, pathogens do not only interact with their hosts but also with its microbiota. Since this fact became only apparent within the last few years more research is needed to reveal all its aspects. A stable foundation for such future work is paved by my recently established germ-free B. germanica breeding system. In this framework it will be particularly important and likewise exciting to perform refaunation experiments with single microbial taxa followed by infections with different pathogens and further transcriptomic analyses to uncover their special tasks in this broad network of interactions

Evidence for reduced immune gene diversity and activity during the evolution of termites

This study was supported by Freie Universität Internal Research Funding and Devtsche Forschungsgemeinschaft (DFG, grant no. MC 436/5-1) to D.P.M. S.H., P.S. and J.S. are supported by ‘EVA4.0’ (no. CZ.02.1.01/0.0/0.0/16_019/0000803), and P.S. and J.S. are supported by CIGA no. 20184306. Y.C. and Z.W. are supported by the National Natural Science Foundation of China (grant no. 31672329).The evolution of biological complexity is associated with the emergence of bespoke immune systems that maintain and protect organism integrity. Unlike the well-studied immune systems of cells and individuals, little is known about the origins of immunity during the transition to eusociality, a major evolutionary transition comparable to the evolution of multicellular organisms from single-celled ancestors. We aimed to tackle this by characterizing the immune gene repertoire of 18 cockroach and termite species, spanning the spectrum of solitary, subsocial and eusocial lifestyles. We find that key transitions in termite sociality are correlated with immune gene family contractions. In cross-species comparisons of immune gene expression, we find evidence for a caste-specific social defence system in termites, which appears to operate at the expense of individual immune protection. Our study indicates that a major transition in organismal complexity may have entailed a fundamental reshaping of the immune system optimized for group over individual defence.Peer reviewe

Eating in a losing cause : limited benefit of modified macronutrient consumption following infection in the oriental cockroach Blatta orientalis

Open Access funding enabled and organized by Projekt DEAL. S.H. was supported by the Chinese Scholarship Council and D.P.M. was supported by a seed-funding Grant provided by the Freie Universität Berlin and grant MC 436/6-1 from the Deutsche Forschungsgemeinschaft (DFG).Background Host–pathogen interactions can lead to dramatic changes in host feeding behaviour. One aspect of this includes self-medication, where infected individuals consume substances such as toxins or alter their macronutrient consumption to enhance immune competence. Another widely adopted animal response to infection is illness-induced anorexia, which is thought to assist host immunity directly or by limiting the nutritional resources available to pathogens. Here, we recorded macronutrient preferences of the global pest cockroach, Blatta orientalis to investigate how shifts in host macronutrient dietary preference and quantity of carbohydrate (C) and protein (P) interact with immunity following bacterial infection. Results We find that B. orientalis avoids diets enriched for P under normal conditions, and that high P diets reduce cockroach survival in the long term. However, following bacterial challenge, cockroaches significantly reduced their overall nutrient intake, particularly of carbohydrates, and increased the relative ratio of protein (P:C) consumed. Surprisingly, these behavioural shifts had a limited effect on cockroach immunity and survival, with minor changes to immune protein abundance and antimicrobial activity between individuals placed on different diets, regardless of infection status. Conclusions We show that cockroach feeding behaviour can be modulated by a pathogen, resulting in an illness-induced anorexia-like feeding response and a shift from a C-enriched to a more P:C equal diet. However, our results also indicate that such responses do not provide significant immune protection in B. orientalis, suggesting that the host’s dietary shift might also result from random rather than directed behaviour. The lack of an apparent benefit of the shift in feeding behaviour highlights a possible reduced importance of diet in immune regulation in these invasive animals, although further investigations employing pathogens with alternative infection strategies are warranted.Peer reviewe

Evidence for reduced immune gene diversity and activity during the evolution of termites

The evolution of biological complexity is associated with the emergence of bespoke immune systems that maintain and protect organism integrity. Unlike the well-studied immune systems of cells and individuals, little is known about the origins of immunity during the transition to eusociality, a major evolutionary transition comparable to the evolution of multicellular organisms from single-celled ancestors. We aimed to tackle this by characterizing the immune gene repertoire of 18 cockroach and termite species, spanning the spectrum of solitary, subsocial and eusocial lifestyles. We find that key transitions in termite sociality are correlated with immune gene family contractions. In cross-species comparisons of immune gene expression, we find evidence for a caste-specific social defence system in termites, which appears to operate at the expense of individual immune protection. Our study indicates that a major transition in organismal complexity may have entailed a fundamental reshaping of the immune system optimized for group over individual defence

Eating in a losing cause: limited benefit of modified macronutrient consumption following infection in the oriental cockroach Blatta orientalis

Background Host–pathogen interactions can lead to dramatic changes in host feeding behaviour. One aspect of this includes self-medication, where infected individuals consume substances such as toxins or alter their macronutrient consumption to enhance immune competence. Another widely adopted animal response to infection is illness-induced anorexia, which is thought to assist host immunity directly or by limiting the nutritional resources available to pathogens. Here, we recorded macronutrient preferences of the global pest cockroach, Blatta orientalis to investigate how shifts in host macronutrient dietary preference and quantity of carbohydrate (C) and protein (P) interact with immunity following bacterial infection. Results We find that B. orientalis avoids diets enriched for P under normal conditions, and that high P diets reduce cockroach survival in the long term. However, following bacterial challenge, cockroaches significantly reduced their overall nutrient intake, particularly of carbohydrates, and increased the relative ratio of protein (P:C) consumed. Surprisingly, these behavioural shifts had a limited effect on cockroach immunity and survival, with minor changes to immune protein abundance and antimicrobial activity between individuals placed on different diets, regardless of infection status. Conclusions We show that cockroach feeding behaviour can be modulated by a pathogen, resulting in an illness-induced anorexia-like feeding response and a shift from a C-enriched to a more P:C equal diet. However, our results also indicate that such responses do not provide significant immune protection in B. orientalis, suggesting that the host’s dietary shift might also result from random rather than directed behaviour. The lack of an apparent benefit of the shift in feeding behaviour highlights a possible reduced importance of diet in immune regulation in these invasive animals, although further investigations employing pathogens with alternative infection strategies are warranted

Evidence for reduced immune gene diversity and activity during the evolution of termites

This dataset contains data from a termite immunity related study described in the paper: “He Shulin, Sieksmeyer Thorben, Che Yanli, Mora M. Alejandra Esparza, Stiblik Petr, Banasiak Ronald, Harrison Mark C., Šobotník Jan, Wang Zongqing, Johnston Paul R. and McMahon Dino P. 2021Evidence for reduced immune gene diversity and activity during the evolution of termitesProc. R. Soc. B.288:20203168.http://doi.org/10.1098/rspb.2020.3168”. The study investigates the evolution of termite molecular immune system: evolution of immune gene family along a constructed phylogeny, different individual immune response between three termite castes, a subsocial cockroach and a non-social cockroach, the caste specific expression of immune genes, different social immune response between a social termite species and a non-social cockroach species. In the first experiment, we de novo sequenced 18 cockroach and termite species, spanning the full spectrum of solitary and social lifestyles, including two solitary cockroach species, two species of subsocial Cryptocercus wood-feeding cockroaches and 14 termite species. We exploited a transcriptomic approach to compare the immune gene repertoire of these sequenced species. In the second experiment, we compared individual immune responses in a solitary cockroach, B. orientalis, a subsocial wood-feeding roach, Cryptocercus meridianus, and each caste of a social termite, Neotermes castaneus, following direct injection with heat-killed microbes. In the third experiment, we explored total gene expression differences between castes without immune challenge. In the fourth experiment, we studied gene expression changes in each caste of N. castaneus following colony exposure to immune-challenged nestmates, and compared these with gene expression changes in the solitary cockroach, B. orientalis, following group exposure to immune-challenged conspecifics. Main results of the experiments are that (1) immune gene families show contractions and expansions during temite evolution; (2) compared with cockroaches, termites showed weak individual immune response; (3) termites have caste-specific constitutive immunity; (4) Compared with cockroach, termite showed a stronger gene expression changes in response to a social immune challenge