Multifaceted Defense against Antagonistic Microbes in Developing Offspring of the Parasitoid Wasp Ampulex compressa (Hymenoptera, Ampulicidae)

Effective antimicrobial strategies are essential adaptations of insects to protect themselves, their offspring, and their foods from microbial pathogens and decomposers. Larvae of the emerald cockroach wasp, Ampulex compressa, sanitize their cockroach hosts, Periplaneta americana, with a cocktail of nine antimicrobials comprising mainly (R)-(-)-mellein and micromolide. The blend of these antimicrobials has broad-spectrum antimicrobial activity. Here we explore the spatio-temporal pattern of deployment of antimicrobials during the development from egg to adult as well as their physico-chemical properties to assess how these aspects may contribute to the success of the antimicrobial strategy. Using gas chromatography/mass spectrometry (GC/MS) we show that larvae start sanitizing their food as soon as they have entered their host to feed on its tissue. Subsequently, they impregnate the cockroach carcass with antimicrobials to create a hygienic substrate for cocoon spinning inside the host. Finally, the antimicrobials are incorporated into the cocoon. The antimicrobial profiles on cockroach and wasp cocoon differed markedly. While micromolide persisted on the cockroaches until emergence of the wasps, solid-phase microextraction sampling and GC/MS analysis revealed that (R)-(-)-mellein vaporized from the cockroaches and accumulated in the enclosed nest. In microbial challenge assays (R)-(-)-mellein in the headspace of parasitized cockroaches inhibited growth of entomopathogenic and opportunistic microbes (Serratia marcescens, Aspergillus sydowii, Metarhizium brunneum). We conclude that, in addition to food sanitation, A. compressa larvae enclose themselves in two defensive walls by impregnating the cocoon and the cockroach cuticle with antimicrobials. On top of that, they use vaporous (R)-(-)-mellein to sanitize the nest by fumigation. This multifaceted antimicrobial defense strategy involving the spatially and temporally coordinated deployment of several antimicrobials in solution and vapor form has apparently evolved to reliably protect the larvae themselves and their food against a broad range of antagonistic microbes.


Methods
To make sure that the whitish colonies growing on agar cubes incubated in the headspace of parasitized cockroaches were S. marcescens and not contaminations, we compared a partial 16S rDNA sequence of these colonies with the red colonies growing on control agar cubes.
We set up an additional bacterial challenge assay as described in the Methods section to obtain bacterial colonies for genetic analyses. For genetic analyses, samples of single colonies from two control and two test agar cubes were picked with sterile toothpicks.
The DNA was extracted with a MasterPure™ Complete DNA and RNA Purification Kit (Epicentre Biotechnologies) following the manufacturer's instructions. The DNA was then used as a template for polymerase chain reaction (PCR) with oligonucleotide primers fD1 (forward) and rP2 (reverse) [1] for the amplification of fragments of the 16S rDNA.

Results
The partial 16S rDNA sequences that we obtained for the red (808 and 810bp) and the whitish (813 and 816bp) bacterial colonies in the bacterial challenge assays were identical ( Figure S3). Furthermore the sequence showed 100% homology with the S. marcescens strain originally isolated from cockroaches in a previous study [2] (GenBank accession number: JX448402) and inoculated on the agar cubes here.

Discussion
The bacteria forming whitish colonies on the agar cubes incubated in the headspace of parasitized cockroaches were genetically identical to the S. marcescens strain forming red colonies on the control agar cubes, and do thus represent decolorized colonies of the originally inoculated bacterium.

Isolation and identification of fungi from cockroaches Methods
To obtain possible antagonistic fungi that might threaten A. compressa offspring during their development we probed incubated nests that failed to produce adult A. compressa wasps and that appeared to be infested by fungi (occurrence of fungal hyphae and/or conidiophores). Probes of fungi were isolated from four infested, parasitized cockroaches by use of sterile forceps. The fungi were inoculated onto Sabouraud-dextrose (SD) agar plates and placed in a conditioning cabinet at 30°C until visible growth of microbes. For the identification of the fungi, single mature conidiophores were seeded on new SD agar plates at least twice to obtain pure strains. From these colonies samples were taken for identification by Sanger sequencing of rDNA internal transcribed spacer regions (ITS) 1 and 2. In addition, morphological characteristics of the cultivated strains were investigated and recorded.
Growing mycelia of the fungi were sampled, ground in liquid nitrogen and the DNA extracted as described above for S. marcescens. The DNA was then used as a template for PCR with oligonucleotide primers ITS1 (forward) and ITS4 (reverse) [3] for the amplification of fragments of rDNA regions ITS1 and ITS2.
PCR reactions and amplicon purification were performed as described above for S. marcescens. The amplicons were subsequently sequenced commercially (Eurofins MWG Operon) with primers ITS1/ITS4. The obtained partial ITS sequences were submitted to the international nucleotide sequence library GenBank (see results for accession numbers) and compared to sequences published in GenBank using the BLAStn algorithm of the National Center of Biotechnology Information (NCBI).

Results
From the infested cockroaches we obtained four fungal isolates. Two of the isolates grew rapidly and exhibited intense yellow-green colonies after four days of incubation. One of these isolates was used for genetic analyses. The partial ITS sequence of this fungal isolate (530bp, GenBank accession number: KF978105]) showed 100% homology with Aspergillus nomius Kurtzman, Horn and Hesseltine ITS sequences (e.g. GenBank accession number: JF824686.1) in the GenBank database. The two other fungal isolates grew more slowly and developed colonies that were variable in color ranging from beige-brown to dark green after five days. The partial ITS sequence of this fungus (497bp, GenBank accession number: KF978104) had 100% homology with an ITS sequence published for Aspergillus sydowii Thom and Church (GenBank accession number: KC253961.1).

Discussion
In this study we isolated and identified the two filamentous fungi A. nomius and A. sydowii from failed A. compressa nests. The ubiquitous fungus A. nomius belongs to the section Flavi of the genus Aspergillus and is a highly aggressive, fast growing, and aflatoxinproducing species. It is known as an opportunistic facultative entomopathogen, and has also been reported as soil fungus, as mold on stored food and as pathogen in humans [4][5][6][7][8]. A. sydowii belongs to the Aspergillus versicolor section and has been isolated from insects [9], soil and air, as well as water [10]. It is known to infest grain cultures [11] and to cause Aspergillosis in various animals from corals to humans [12,13].