Applying microsatellite DNA markers in population genetic studies of the pest moth Helicoverpa armigera is subject to numerous technical problems, such as the high frequency of null alleles, occurrence of size homoplasy, presence of multiple copies of flanking sequence in the genome and the lack of PCR amplification robustness between populations. To overcome these difficulties, we developed exon-primed intron-crossing (EPIC) nuclear DNA markers for H. armigera based on ribosomal protein (Rp) and the Dopa Decarboxylase (DDC) genes and sequenced alleles showing length polymorphisms. Allele length polymorphisms were usually from random indels (insertions or deletions) within introns, although variation of short dinucleotide DNA repeat units was also detected. Mapping crosses demonstrated Mendelian inheritance patterns for these EPIC markers and the absence of both null alleles and allele ‘dropouts’. Three examples of allele size homoplasies due to indels were detected in EPIC markers RpL3, RpS6 and DDC, while sequencing of multiple individuals across 11 randomly selected alleles did not detect indel size homoplasies. The robustness of the EPIC-PCR markers was demonstrated by PCR amplification in the related species, H. zea, H. assulta and H. punctigera.

The melanization of Dirofilaria immitis microfilariae in Armigeres subalbatus haemolymph in vitro is a two-step process. Firstly, the microfilariae are encased in a transparent capsule, then the capsule material is melanized later. Benzamidine HCl and p-amidinophenylmethanesulfonyl fluoride, both serine protease inhibitors, inhibited the deposition of the transparent capsule material and melanization. Diethyldithiocarbamate, a phenoloxidase inhibitor, did not prevent the deposition of the transparent capsule material but it did inhibit melanization. m-Hydroxybenzylhydrazine, a dopa decarboxylase inhibitor, had no inhibitory action on the deposition of the transparent capsule material. However, the time for the transparent capsule to become melanized was delayed due to the presence of m-hydroxybenzylhydrazine in the test system. Immunocytochemical localization showed that prophenoloxidase and/or phenoloxidase was present in the transparent capsule material. These results suggest (1) that prophenoloxidase cascade is essential in the melanization of microfilariae in mosquitoes and its putative activation behaviour is similar to that of other insects and (2) that the deposition of transparent capsule material is a result of the activation of prophenoloxidase by serine proteases and the melanization of the transparent capsule material is due to the presence of active phenoloxidase in capsule.