FAMILY-SPECIFIC COX1 PRIMERS FOR IDENTIFYING APHIDS FROM DEGRADED SAMPLES

Aphids (Hemiptera: Aphididae) are significant agricultural pests and vectors of plant viruses, making accurate species identification an essential component of pest management and ecological studies. However, DNA degradation in preserved aphid samples presents a challenge for reliable molecular identification. This study aimed to develop family-specific primers for amplifying and sequencing the COX1 barcode region for species identification of old aphid samples. Aphid samples were collected from bucket traps in Christiana, South Africa, between 2006 and 2008, representing 31 species across 21 genera. Morphological identification was conducted initially, and the samples were then stored for long-term preservation in ethanol, prior to primer testing. Universal arthropod primers (LCO1490/HCO2198 and LepF/LepR) failed to amplify the samples because they are old and likely have degraded DNA, highlighting the need to amplify smaller regions of the COX1 gene due to DNA degradation. Family-specific primers were designed based on publicly available mitochondrial COX1 sequences from Aphididae, targeting short overlapping fragments (162–178 bp) of the COX1 gene to reconstruct the complete barcode region. A 294 bp mini-barcode was identified, providing reliable species-level resolution for five aphid species: Acyrthosiphon kondoi, Acyrthosiphon pisum, Macrosiphum euphorbiae, Metopolophium dirhodum, and Tetraneura fusiformis. Species identifications were validated against a curated reference database, achieving high taxonomic resolution. The primers successfully amplified DNA from moderately degraded specimens but were ineffective for severely degraded samples due to DNA fragmentation. This study demonstrates the utility of family-specific primers and the mini-barcode approach for species identification of old aphid samples, offering a robust framework for molecular taxonomy and advancing aphid species identification for ecological research and pest management.