A switch from constitutive chemical defense to inducible innate immune responses in the invasive ladybird Harmonia axyridis

The harlequin ladybird, Harmonia axyridis, has emerged as a model species for invasion biology, reflecting its remarkable capacity to outcompete native ladybird species when introduced into new habitats. This ability may be associated with its prominent resistance to pathogens and intraguild predation. We recently showed that the constitutive antibacterial activity present in the haemolymph of H. axyridis beetles can be attributed to the chemical defence compound harmonine. Here, we demonstrate that H. axyridis differs from other insects, including the native ladybird Coccinella septempunctata, by reducing rather than increasing the antimicrobial activity of its haemolymph following the injection of bacteria. However, both species produce new or more abundant proteins in the haemolymph, indicating that bacterial challenge induces innate immune responses associated with the synthesis of immunity-related proteins. Our results suggest that H. axyridis beetles can switch from constitutive chemical defence to inducible innate immune responses, supporting hypothesis that inducible antimicrobial peptides protect host beetles against pathogens that survive constitutive defences. These alternative antimicrobial defence mechanisms may reflect a trade-off resulting from fitness-related costs associated with the simultaneous synthesis of harmonine and antimicrobial peptides/proteins

Next-generation sequencing analysis of the Tineola bisselliella larval gut transcriptome reveals candidate enzymes for keratin digestion

The clothes moth Tineola bisselliella is one of a few insects that can digest keratin, leading to the destruction of clothing, textiles and artwork. The mechanism of keratin digestion is not yet fully understood, partly reflecting the lack of publicly available genomic and transcriptomic data. Here we present a high-quality gut transcriptome of T. bisselliella generated from larvae reared on keratin-rich and keratin-free diets. The overall transcriptome consists of 428,221 contigs that were functionally annotated and screened for candidate enzymes involved in keratin utilization. As a mechanism for keratin digestion, we identified cysteine synthases, cystathionine β-synthases and cystathionine γ-lyases. These enzymes release hydrogen sulfite, which may reduce the disulfide bonds in keratin. The dataset also included 27 differentially expressed contigs with trypsin domains, among which 20 were associated with keratin feeding. Finally, we identified seven collagenases that were upregulated on the keratin-rich diet. In addition to this enzymatic repertoire potentially involved in breaking down keratin, our analysis of poly(A)-enriched and poly(A)-depleted transcripts suggested that T. bisselliella larvae possess an unstable intestinal microbiome that may nevertheless contribute to keratin digestion

MMPs Regulate both Development and Immunity in the Tribolium Model Insect

BACKGROUND: Matrix metalloproteinases (MMPs) are evolutionarily conserved and multifunctional effector molecules in development and homeostasis. In spite of previous, intensive investigation in vitro and in cell culture, their pleiotrophic functions in vivo are still not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We show that the genetically amenable beetle Tribolium castaneum represents a feasible model organism to explore MMP functions in vivo. We silenced expression of three insect-type Tribolium MMP paralogs and their physiological inhibitors, TIMP and RECK, by dsRNA-mediated genetic interference (RNAi). Knock-down of MMP-1 arrested development during pupal morphogenesis giving phenotypes with altered antennae, compound eyes, wings, legs, and head. Parental RNAi-mediated knock-down of MMP-1 or MMP-2 resulted in larvae with non-lethal tracheal defects and with abnormal intestines, respectively, implicating additional roles of MMPs during beetle embryogenesis. This is different to findings from the fruit fly Drosophila melanogaster, in which MMPs have a negligible role in embryogenesis. Confirming pleiotrophic roles of MMPs our results also revealed that MMPs are required for proper insect innate immunity because systemic knock-down of Tribolium MMP-1 resulted in significantly higher susceptibility to the entomopathogenic fungus Beauveria bassiana. Moreover, mRNA levels of MMP-1, TIMP, and RECK, and also MMP enzymatic activity were significantly elevated in immune-competent hemocytes upon stimulation. To confirm collagenolytic activity of Tribolium MMP-1 we produced and purified recombinant enzyme and determined a similar collagen IV degrading activity as observed for the most related human MMP, MMP-19. CONCLUSIONS/SIGNIFICANCE: This is the first study, to our knowledge, investigating the in vivo role of virtually all insect MMP paralogs along with their inhibitors TIMP and RECK in both insect development and immunity. Our results from the Tribolium model insect indicate that MMPs regulate tracheal and gut development during beetle embryogenesis, pupal morphogenesis, and innate immune defense reactions thereby revealing the evolutionarily conserved roles of MMPs

Comparative transcriptomics in three ladybird species supports a role for immunity in invasion biology

The spread of the invasive harlequin lady bird (Harmonia axyridis) in Europe is accompanied by the decline of the native and non-invasive two-spotted lady bird (Adalia bipunctata). Here we show that microsporidia carried by H. axyridis can kill A. bipunctata following the oral uptake of spores, suggesting that their horizontal transmission via intraguild predation may help the invader to outcompete its native competitor. The native seven-spotted lady bird (Coccinella septempunctata) is thought to be less susceptible both to the spread of H. axyridis and to its microsporidia. To investigate whether the distinct levels of pathogen susceptibility in these three ladybird species are determined by their immune systems, we compared the immunity-related transcriptomes of untreated beetles and beetles challenged with suspensions of bacteria and yeast. We found that H. axyridis carries three and four times as many genes encoding antimicrobial peptides representing the attacin, coleoptericin and defensin families than C. septempunctata and A. bipunctata, respectively. Gene expression studies following the injection of bacteria and yeasts into beetles revealed that members of these three antimicrobial peptide families are also induced more strongly in H. axyridis than C. septempunctata or A. bipunctata. Our results therefore support the hypothesis that a superior immune system promotes the performance of invasive species

Der Asiatische Marienkäfer als Modell: Invasiv durch biologische und chemische Waffen

The harlequin ladybird Harmonia axyridis has been established as a promined model in invasion biology because it particularly suited to test hypotheses explaining the successful spread of some introduced species. Recent research elucidated that H. axyridis carries parasites which can infect native ladybird species if the feed on its eggs or larvae within intraguild predation. Besides these “bioweapons” H. axyridis accumulates small molecules in its hemolymph which provide as chemical weapons protection against pathogens and predators. The application of state of the art genetic tools will help to elucidate adaptations mediating invasive success at the molecular level

Symbiont‐mediated chemical defense in the invasive ladybird Harmonia axyridis

The volatile alkylpyrazines methyl‐ and methoxypyrazines (MPs) present in the reflex bleeds of coccinellid beetles such as the harlequin ladybird beetle Harmonia axyridis are important semiochemicals that function in antipredatory defense behavior. Pyrazines have also been coadapted from a primarily defensive role into pheromones that function in intraspecific communication, attraction, and aggregation behavior. However, the biosynthesis of MPs in ladybird beetles is poorly understood. Here, we tested the hypothesis that MPs could be produced by microbial symbionts in H. axyridis, which generates four different MPs. The evaluation of tissue‐specific MP production showed that MP concentrations were highest in the gut tissue and hemolymph of the beetles rather than the fat body tissue as the presumed site of MP biosynthesis. Furthermore, manipulation of gut microbiota by antibiotic‐containing diets resulted in a lower MP content in adult beetles. The analysis of the bacterial community of the digestive tract revealed the presence of bacteria of the genera Serratia and Lactococcus which are reportedly able to produce MPs. In line with the known diet‐dependent production of MP in H. axyridis, we determined that the presence or relative abundance of some of the potential MP producers (Enterococcus and Staphylococcus) is also diet‐dependent. We hypothesize a potential role of the microbiota in MP production in H. axyridis as a possible example for outsourcing the synthesis of ecologically important semiochemicals to its gut bacteria

Invasive harlequin ladybird carries biological weapons against native competitors

Invasive species that proliferate after colonizing new habitats have a negative environmental and economic impact. The reason why some species become successful invaders, whereas others, even closely related species, remain noninvasive is often unclear. The harlequin ladybird Harmonia axyridis, introduced for biological pest control, has become an invader that is outcompeting indigenous ladybird species in many countries. Here, we show that Harmonia carries abundant spores of obligate parasitic microsporidia closely related to Nosema thompsoni. These microsporidia, while not harming the carrier Harmonia, are lethal pathogens for the native ladybird Coccinella septempunctata. We propose that intraguild predation, representing a major selective force among competing ladybird species, causes the infection and ultimate death of native ladybirds when they feed on microsporidia-contaminated Harmonia eggs or larvae

The maternal transfer of bacteria can mediate trans-generational immune priming in insects

Parents invest in their offspring by preparing them for defense against pathogens and parasites that only the parents have encountered, a phenomenon known as trans-generational immune priming. We investigated the underlying mechanism using the established lepidopteran model host Galleria mellonella. When larvae were fed with non-pathogenic bacteria, or the entomopathogenic species Pseudomonas entomophila and Serratia entomophila, the activity of lysozyme and phenoloxidase increased in the hemolymph, and immunity-related genes encoding antibacterial proteins such as gloverin were induced. Remarkably, the ingestion of bacteria by female larvae resulted in the differential expression of immunity-related genes in the eggs subsequently laid by the same females, providing evidence for trans-generational immune priming in G. mellonella. To determine the fate of these ingested microbes, the larval diet was supplemented with bacteria carrying a fluorescent label. We observed these bacteria crossing the midgut epithelium, their entrapment within nodules in the hemocoel, their accumulation within the ovary, and ultimately their deposition in the eggs. Therefore, we propose that trans-generational immune priming in Lepidoptera can be mediated by the maternal transfer of bacteria or bacterial fragments to the developing eggs

Evolutionary ecology of microsporidia associated with the invasive ladybird Harmonia axyridis

Invasive species are characterized by the rapid growth and spread of their populations after establishing a foothold in new habitats, and there are now many examples of such species negatively affecting biodiversity and the economy. It is unclear why some species can become successful invaders, whereas most (even if closely related) remain noninvasive. We previously proposed a hypothesis that parasites associated with invading species can promote their invasive success if they are harmless toward the invaders but harmful to their competitors and/or predators in the newly colonized habitat. Here we discuss whether microsporidia that have recently been discovered in the invasive ladybird Harmonia axyridis contribute to its invasive success. We show that all H. axyridis beetles sourced from diverse collection sites all over the world carry abundant microsporidia. This suggests that both native and invasive H. axyridis populations are associated with these tolerated parasites, which were likely to have existed in native populations before expansion rather than being acquired in newly colonized areas. We describe the pathogenesis of the microsporidia during different developmental stages of H. axyridis and we address the possibility that the predation of its infected eggs and larvae by competing native ladybird species may lead to their infection and ultimately to their decline. Finally, we discuss our initial hypothesis: microsporidia that are tolerated by an invasive vector insect can be active against susceptible native competitors and/or predator species