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Invasive Harlequin Ladybird Carries Biological Weapons Against Native Competitors

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Science  17 May 2013:
Vol. 340, Issue 6134, pp. 862-863
DOI: 10.1126/science.1234032

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.

Abstract

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.

Human activities, particularly international trade, promote the spread of invasive species that cause extensive economic losses and negatively affect native species. Several factors can play a role in the invasive success of such species, including the lack of predators, short generation times, and the ability to disperse rapidly and adapt easily to new habitats (1, 2). However, the principles that allow some species to become successful invaders, whereas most (even if closely related) do not, remain poorly understood (3). Invaders can be released from native, coevolved pathogens, but they face other pathogens in their new environments, suggesting that the ability to mount strong antimicrobial defenses may promote invasive success (4). Yet enhanced immunity can be costly and, therefore, can be traded off against other traits such as growth and reproduction (5, 6). We used the harlequin ladybird Harmonia axyridis (a native species in central Asia) as a model to explore the potential role of immunity in invasion biology. This species has been introduced into many countries as a biological control agent against aphids and other insect pests but is now causing severe problems because it successfully outcompetes native ladybird species in many areas (7).

We recently showed that, in contrast to native ladybird species in Europe, the Harmonia hemolymph contains strong and constitutive antibacterial activity throughout development. We attributed this activity to harmonine, a secondary metabolite that accumulates to high levels in the hemolymph. The broad-spectrum activity of this alkaloid compound is demonstrated by its ability to inhibit even human pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum, making it a promising lead for the development of new anti-infective drugs (8). Constitutive harmonine activity may help Harmonia to deal with pathogens encountered in new habitats, whereas native ladybirds are more susceptible to infection (9).

Independently, harmonine has been proposed as a chemical defense compound protecting Harmonia eggs and larvae from predation by native ladybird species (10). Kajita et al. reported that the ingestion of Harmonia eggs by native Coccinella septempunctata beetles caused mortality, but the reciprocal situation was nonlethal. In agreement with our previous report (8), they detected high concentrations of harmonine in Harmonia eggs and concluded that this compound protects the invasive ladybird from intraguild predation (10), which is a major selective force among competing ladybird species (11).

Here, we report that the injection of Harmonia hemolymph—but not synthetic harmonine alone, even in high concentrations (fig. S1)—can kill Coccinella beetles, making it unlikely that the mortality caused by feeding on Harmonia eggs is caused by the presence of harmonine. It is therefore apparent that the mortality in Coccinella beetles is caused by another component found in the hemolymph of Harmonia.

Light microscopy revealed the presence of abundant microsporidia among the hemocytes in Harmonia hemolymph (Fig. 1A) (see supplementary materials and methods). Microsporidia are obligate parasites that replicate within eukaryotic cells after penetrating the plasma membrane with an extruded polar tube. Semithin sections of hemolymph tissue confirmed the presence of microsporidia, among which some exhibited extruded polar tubes (Fig. 1B). We used scanning electron microscopy to document the high abundance of microsporidia in the hemolymph of Harmonia (Fig. 1C). Despite the abundance of spores, we did not find any Harmonia beetles that were killed by the parasites in our rearing. The low physiological activity of the microsporidia is further supported by the absence of microsporidial gene expression when analyzing the transcriptome of Harmonia eggs and beetles (12). These data suggest that the microsporidia are present in a physiologically inactive spore stage and do not harm their host, perhaps because it has acquired tolerance or resistance.

Fig. 1 Microscopy studies of microsporidia.

(A) Light microscopy image showing the high concentration of microsporidia (small objects) between the larger hemocytes (arrow) in Harmonia hemolymph. Scale bar, 50 μm. (B) Semithin sections of hemolymph tissue confirm the presence of microsporidia, among which some exhibit extruded polar tubes (circles). Extrusion of polar tubes occurred during fixation. Scale bar, 20 μm. (C) Scanning electron microscopy image of microsporidia in the Harmonia hemolymph, showing the high load of spores. Scale bar, 20 μm. (D) Light microscopy image showing microsporidia (arrows) between yeastlike cells and larger hemocytes in dying Coccinella beetles 7 days postinoculation. Scale bar, 20 μm.

We identified the microsporidia by amplifying the small-subunit ribosomal RNA (rRNA) genes using a variety of previously described primer sets (13), resulting in the specific amplification of the partial microsporidial 16S rRNA gene. The determined sequence identity (≥99%) placed the Harmonia-associated microsporidia within the Nosema/Vairimorpha clade, with Nosema thomsoni as the closest relative (14). This assay confirmed the presence of Nosema-like microsporidia in all Harmonia beetles of all populations sampled, as well as in eggs and larvae, suggesting vertical transmission. All attempts to coculture the microsporidia with different insect cell lines (Sf9, Sf21, Drosophila S2, High Five) failed.

Given that (i) Coccinella beetles are killed by feeding on Harmonia eggs and larvae, but the reciprocal situation is not lethal (10), and (ii) harmonine does not affect Coccinella beetles, even at high concentrations, we propose that native ladybird species may be lethally infected by the microsporidia carried by Harmonia when they feed on its eggs and larvae. To test this hypothesis, we collected hemolymph samples from Harmonia and isolated the microsporidia by repeated centrifugation and washing steps. We divided the purified microsporidia into two portions, one of which was heat-inactivated and used for control injections.

All Coccinella beetles injected with live microsporidia isolated from Harmonia died within 2 weeks (Fig. 2), whereas the majority of control beetles injected with either the heat-inactivated microsporidia or the buffer alone survived. Control injections with cell-free hemolymph samples from Harmonia lacking microsporidia and hemocytes did not result in enhanced mortality (fig. S4). This observation and the analysis of injected samples by SDS–polyacrylamide gel electrophoresis (fig. S2) and mass spectrometry ruled out the possibility that the mortality was caused by thermolabile toxins in the hemolymph of Harmonia. Further, we determined the presence of microsporidia in dying Coccinella, but not in control beetles (Fig. 1D and fig. S3). We concluded that the microsporidia carried by Harmonia were lethal but required some time to infect and replicate within Coccinella.

Fig. 2 Survival after injection.

Coccinella beetle survival rate (y axis) analysis after transfer of microsporidia isolated from the hemolymph of Harmonia (Ha-Msp in Cs), calculated by Kaplan Meier survival analysis log-rank test. Coccinella beetles were injected with living microsporidia (solid line). Control injections with either heat-inactivated microsporidia (heated Ha-Msp) isolated from Harmonia (dashed line) or PBS alone (dotted line) showed statistically significant differences (P < 0.05), indicating that mortality of Coccinella is predominantly caused by living microsporidia. Circles indicate where animals have been taken out of the experiment to control the presence of microsporidia in the hemolymph. Cs, Coccinella septempunctata; PBS, phosphate-buffered saline.

The high abundance of tolerated microsporidia in Harmonia hemolymph and their ability to kill Coccinella beetles support our hypothesis that these parasites contribute to the dominance of Harmonia over native species. Obviously, native ladybirds such as Coccinella do not share with Harmonia the ability to suppress microsporidial replication. It remains to be seen whether or not harmonine (8) and/or the tremendous spectrum of antimicrobial peptides discovered in Harmonia (12) contribute to its tolerance or resistance against microsporidia. Our data also provide a candidate mechanism to explain why the decline in native ladybird numbers is associated with intraguild predation (11). The presence of microsporidia in Harmonia may function like a biological weapon, in accordance with the novel weapons theory (15).

Supplementary Materials

www.sciencemag.org/cgi/content/full/340/6134/862/DC1

Materials and Methods

Figs. S1 to S4

Tables S1 to S3

References (16, 17)

References and Notes

  1. The partial microsporidial 16S rRNA sequence has been deposited in the public database GenBank under accession no. KC596023.
  2. Acknowledgments: We acknowledge project funding provided by the excellence initiative of the Hessian Ministry of Science and Art via the LOEWE research focus "Insect Biotechnology." We thank J. Rolff for critical reading and R. M. Twyman for editing of the manuscript.
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