We established a protoplast-based system to transfer DNA to Knufia petricola strain A95, a melanised rock-inhabiting microcolonial fungus that is also a component of a model sub-aerial biofilm (SAB) system. To test whether the desiccation resistant, highly melanised cell walls would hinder protoplast formation, we treated a melanin-minus mutant of A95 as well as the type-strain with a variety of cell-degrading enzymes. Of the different enzymes tested, lysing enzymes from Trichoderma harzianum were most effective in producing protoplasts. This mixture was equally effective on the melanin-minus mutant and the type-strain. Protoplasts produced using lysing enzymes were mixed with polyethyleneglycol (PEG) and plasmid pCB1004 which contains the hygromycin B (HmB) phosphotransferase (hph) gene under the control of the Aspergillus nidulans trpC. Integration and expression of hph into the A95 genome conferred hygromycin resistance upon the transformants. Two weeks after plating out on selective agar containing HmB, the protoplasts developed cell-walls and formed colonies. Transformation frequencies were in the range 36 to 87 transformants per 10 μg of vector DNA and 106 protoplasts. Stability of transformation was confirmed by sub-culturing the putative transformants on selective agar containing HmB as well as by PCR-detection of the hph gene in the colonies. The hph gene was stably integrated as shown by five subsequent passages with and without selection pressure.

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.

The evolution of biological complexity is associated with the emergence of bespoke immune systems that maintain and protect organism integrity. Unlike the well studied immunity at the cell and individual level, 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 tackle this by characterizing the immune gene repertoire of 18 cockroach and termite species, spanning the spectrum of solitary, subsocial and eusocial lifestyles. We identified five significant immune gene family contractions and one immune gene family expansion along the spine of a time-calibrated phylogeny, correlating with key transitions in termite sociality. In cross-species comparisons of immune gene expression, we find that termites appear to have evolved a caste-specific social defense system at the expense of individual immune protection. Our study indicates that a major transition in organismal complexity entailed a fundamental reshaping of the immune system optimized for group over individual defense.

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, minerals or secondary compounds or alter their macronutrient consumption to enhance immune competence. Another aspect includes illness-induced anorexia, which is a general mechanism adopted by animals following infection. Anorexia 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 interact with immunity following sublethal bacterial infection. We compared the survival of uninfected and infected individuals on diets enriched with carbohydrate (C) or protein (P) and conducted food-choice experiments after challenging hosts with the common entomopathogenic soil bacterium Pseudomonas entomophila. We then carried out a quantitative proteomic analysis and an antimicrobial activity assay of hemolymph from infected individuals that had been restricted to diets with defined macronutrient compositions. We find that diets enriched for P reduce cockroach survival. However, following immune challenge by P. entomophila, cockroaches significantly reduced their overall nutrient intake, particularly of carbohydrates, and increased the ratio of protein (P:C) consumed. Surprisingly, these behavioural shifts did not significantly improve cockroach immunity or survival, with negligible differences in immune protein abundance and antimicrobial activity between infected individuals placed on P- or C- rich diets.

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.