COI Gene-based molecular taxonomy of scale insect pests in tropical fruit orchards of Selangor, Malaysia

Introduction

Leading authorities in coccidology have consistently emphasized that molecular approaches are indispensable for achieving stable taxonomic frameworks within the scale insects (Hemiptera: Coccoidea), a group characterized by extreme morphological conservatism, sexual dimorphism, and neotenic body plan reduction that severely limits the utility of traditional diagnostic characters [1,2] . Scale insects rank among the most damaging pest groups in tropical fruit production systems across Southeast Asia, causing direct damage through phloem feeding and indirect losses through honeydew-associated sooty mould development and the transmission of plant pathogens [3] . Malaysia is a significant producer of tropical fruits including durian, rambutan, mangosteen, and starfruit, with Selangor serving as a major production and distribution hub [4] . The scale insect pest complex on these fruit crops includes armoured scales (Diaspididae), soft scales (Coccidae), and mealybugs (Pseudococcidae), each requiring different management approaches [2,5] . DNA barcoding using the COI gene provides a standardized molecular approach for scale insect identification that overcomes the limitations of morphological taxonomy [6,7] . This research aimed to characterize the molecular taxonomy of scale insect pests in tropical fruit orchards of Selangor using COI barcoding. The objectives were to: (i) identify scale insect species; (ii) quantify genetic divergence; (iii) reconstruct phylogenetic relationships; and (iv) assess the host associations of identified species across different fruit crop systems.

Materials and Methods

Materials

Methods

Genomic DNA was extracted from individual scale insects using the DNeasy Blood and Tissue Kit (Qiagen, Germany) with extended proteinase K digestion and mechanical disruption of the waxy shield. COI fragments were amplified using LCO1490/HCO2198 primers [8] . PCR reactions were conducted in 25 μL volumes under cycling conditions of 94 °C for min, 35 cycles of 94 °C for 30 s, 50 °C for 45 s, and 72 °C for 1 min, with final extension at72 °C for 10 min. Products were sequenced bidirectionally, edited in BioEdit v7.2.5, aligned in MEGA 11 [9] , and compared against BOLD and GenBank [10] . K2P distances were calculated [11] , and neighbor-joining trees constructed with 1, 000 bootstrap replicates [12] .

Results

Of 188 scale insect specimens collected, 163 yielded highquality COI sequences (86.7% success rate). Four scale insect species representing two families (Diaspididae and Coccidae) were identified through database comparisons. destructor was the most generalist species, occurring on all four fruit crop types, while U. yanonensis showed the strongest host preference for starfruit orchards.

Discussion

The molecular identification of four scale insect species in Selangor tropical fruit orchards establishes the first comprehensive barcode-based inventory for this pest group in Malaysian fruit production systems. The dominance of A. destructor (32.4%) is consistent with its globally recognized status as one of the most polyphagous armoured scale species, with recorded host plants spanning over 100 families [1,3] . The relatively lower sequencing success rate (86.7%) compared to softer-bodied insect groups reflects the challenges of DNA extraction from scale insects with heavily sclerotized or waxy body coverings [2,6] . The diaspidid species, which possess rigid armoured covers, showed lower individual extraction success than the softbodied coccid species, suggesting that optimized mechanical disruption protocols should be developed for routine molecular diagnostics of armoured scales. The interspecific distances (10.5-19.8%) substantially exceed the 3% species delimitation threshold, confirming unambiguous molecular separation [5,7] . The phylogenetic topology is consistent with current molecular systematic treatments of the Coccoidea, reinforcing the reliability of COI barcoding for species-level identification within this taxonomically challenging group [1,2] . The host association patterns identified have practical implications for orchard-specific pest management. The generalist nature of A. destructor suggests that control programmes in mixed-fruit production landscapes must account for potential cross-infestation between adjacent orchards, while the apparent host specialization of U. yanonensis on starfruit may allow more targeted interventions in starfruit monocultures [3,4] .

Quarantine Diagnostics

Several of the identified scale insect species are regulated quarantine pests in international fruit trade. The COI reference sequences generated provide a foundation for developing molecular diagnostic protocols applicable at Malaysian phytosanitary inspection facilities, enabling rapid and unambiguous identification of intercepted specimens on export fruit consignments [2,5] .

Biological Control Integration

Accurate molecular identification of scale insect species is prerequisite for selecting appropriate parasitoid wasps and predatory insects for biological control programmes. The species-level resolution achieved through COI barcoding enables targeted deployment of natural enemies with demonstrated host specificity against individual scale insect taxa in Malaysian fruit orchards [3,4] . The parasitoid wasp Aphytis lingnanensis, for instance, demonstrates high efficacy against diaspidid scales but is ineffective against coccid species, underscoring the importance of molecular identification for biological control decision-making.

Economic Impact Assessment

The host association data generated in this research provide a foundation for estimating species-specific economic impacts across different fruit crop systems in Selangor. The generalist feeding behavior of A. destructor, occurring across all four surveyed fruit types, suggests that economic impact assessments should account for cross-crop damage potential in mixed orchard landscapes. Conversely, the apparent specialization of U. yanonensis on starfruit allows for more precise damage attribution and targeted costbenefit analyses of species-specific control interventions in starfruit production systems [1,3,4] .

Conclusion

Scale insect pests represent a persistent challenge for tropical fruit production in Selangor, Malaysia, yet

Acknowledgement

Acknowledgements N.A.B.R. designed the research and drafted the manuscript; A.F.B.M.Y. conducted field sampling; S.N.B.A. performed molecular analyses. This research was funded by the Malaysian Agricultural Research and Development Institute. The authors acknowledge the Selangor Institute of Tropical Sciences for laboratory facilities and orchard access.

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