Henrike Schmidtberg

Invasive Harlequin Ladybird Carries Biological Weapons Against Native Competitors

Surprise Attack Humans conduct the largest ecological experiment ever by continually moving species between continents. For example, the harlequin ladybird beetle, native to Asia, has become highly invasive in many regions after being introduced for biological control, but we do not understand why this species should so readily outcompete native ladybirds. Vilcinskas et al. (p. 862; see the Perspective by Reynolds) show that harlequin beetles have parasitic microsporidia within their hemolymph, which are fatal to other ladybird beetles that prey on harlequin beetle eggs and larvae. Harlequin beetles thus have an innate advantage over species that are otherwise equivalent in their abilities, but this sort of competitive advantage can be hard to spot. Invasive ladybugs harbor microsporidia that kill nonresistant beetles contributing to the original residents’ decline. [Also see Perspective by Reynolds] 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.

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.

Protected by Fumigants: Beetle Perfumes in Antimicrobial Defense

Beetles share with other eukaryotes an innate immune system that mediates endogenous defense against pathogens. In addition, larvae of some taxa produce fluid exocrine secretions that contain antimicrobial compounds. In this paper, we provide evidence that larvae of the brassy willow leaf beetle Phratora vitellinae constitutively release volatile glandular secretions that combat pathogens in their microenvironment. We identified salicylaldehyde as the major component of their enveloping perfume cloud, which is emitted by furrow-shaped openings of larval glandular reservoirs and which inhibits in vitro the growth of the bacterial entomopathogen Bacillus thuringiensis. The suggested role of salicylaldehyde as a fumigant in exogenous antimicrobial defense was confirmed in vivo by its removal from glandular reservoirs. This resulted in an enhanced susceptibility of the larvae to infection with the fungal entomopathogens Beauveria bassiana and Metarhizium anisopliae. Consequently, we established the hypothesis that antimicrobial defense in beetles can be expanded beyond innate immunity to include external disinfection of their microenvironment, and we report for the first time the contribution of fumigants to antimicrobial defense in animals.

Harmonine, a defence compound from the harlequin ladybird, inhibits mycobacterial growth and demonstrates multi-stage antimalarial activity

The harlequin ladybird beetle Harmonia axyridis has been introduced in many countries as a biological control agent, but has become an invasive species threatening the biodiversity of native ladybirds. Its invasive success has been attributed to its vigorous resistance against diverse pathogens. This study demonstrates that harmonine ((17R,9Z)-1,17-diaminooctadec-9-ene), which is present in H. axyridis haemolymph, displays broad-spectrum antimicrobial activity that includes human pathogens. Antibacterial activity is most pronounced against fast-growing mycobacteria and Mycobacterium tuberculosis, and the growth of both chloroquine-sensitive and -resistant Plasmodium falciparum strains is inhibited. Harmonine displays gametocytocidal activity, and inhibits the exflagellation of microgametocytes and zygote formation. In an Anopheles stephensi mosquito feeding model, harmonine displays transmission-blocking activity.

A switch from constitutive chemical defence 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.

Translocation of bacteria from the gut to the eggs triggers maternal transgenerational immune priming in Tribolium castaneum

Invertebrates can be primed to enhance their protection against pathogens they have encountered before. This enhanced immunity can be passed maternally or paternally to the offspring and is known as transgenerational immune priming. We challenged larvae of the red flour beetle Tribolium castaneum by feeding them on diets supplemented with Escherichia coli, Micrococcus luteus or Pseudomonas entomophila, thus mimicking natural exposure to pathogens. The oral uptake of bacteria induced immunity-related genes in the offspring, but did not affect the methylation status of the egg DNA. However, we observed the translocation of bacteria or bacterial fragments from the gut to the developing eggs via the female reproductive system. Such translocating microbial elicitors are postulated to trigger bacterial strain-specific immune responses in the offspring and provide an alternative mechanistic explanation for maternal transgenerational immune priming in coleopteran insects.

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.

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

ABSTRACT The spread of the invasive harlequin ladybird (Harmonia axyridis) in Europe is accompanied by the decline of the native and non‐invasive two‐spotted ladybird (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 ladybird (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. Graphical abstract Figure. No Caption available. HighlightsOral uptake of microsporidia carried by the invasive ladybird Harmonia axyridis can kill the native ladybird Adalia bipunctata.Genes encoding antimicrobial peptides are expanded in H. axyridis relative to Adalia bipunctata and Coccinella septempuncata.Inducible levels of some antimicrobial peptides were higher in H. axyridis compared to A. bipunctata and C. septempuncata.

Expression and characterization of a recombinant i-type lysozyme from the harlequin ladybird beetle Harmonia axyridis.

Lysozymes are enzymes that destroy bacterial cell walls by hydrolysing the polysaccharide component of peptidoglycan. In insects, there are two classes of lysozymes, the c‐type with muramidase activity and the i‐type whose prototypical members from annelids and molluscs possess both muramidase and isopeptidase activities. Many insect genes encoding c‐type and i‐type lysozymes have been identified during genome and transcriptome analyses, but only c‐type lysozymes have been functionally characterized at the protein level. Here we produced one of five i‐type lysozymes represented in the immunity‐related transcriptome of the invasive harlequin ladybird beetle Harmonia axyridis as recombinant protein. This was the only one containing the serine and histidine residues that are thought to be required for isopeptidase activity. This i‐type lysozyme was recombinantly expressed in the yeast Pichia pastoris, but the purified protein was inactive in both muramidase and isopeptidase assays. Transcription and immunofluorescence analysis revealed that this i‐type lysozyme is produced in the fat body but is not inducible by immune challenge. These data suggest that i‐type lysozymes in insects may have acquired novel and as yet undetermined functions in the course of evolution.