Diagnostic puzzle of Anopheles species using morphological vs. molecular approach: a case study in Tripura, a hyperendemic malaria state, Northeast India.

The North-East India Region (NER), an area with several malaria-endemic pockets, differs from the rest of India in vector composition, with An. fluviatilis as the only common major vector, mostly reported during winter in NER but perennially in mainland India. However, in most cases, the most common method of morphological identification has been used, with few studies comparing both. During our longitudinal (2019-2024) entomological study in Tripura, a major malaria-contributing NER State, morphological techniques identified 9 An. fluviatilis collected during winter months, along with 500+ An. minimus. However, molecular investigations confirmed An. fluviatilis to be An. minimus, with 3 new haplotypes. Previously, An. fluviatilis identified morphologically and cytotaxonomically from another state of NER and was later molecularly shown to be a morphological variant of An. minimus. Due to hypermelanization, absence of presector pale spots on the wing and palpal banding pattern, An. minimus earlier probably during winter was misidentified as An. fluviatilis in NER while it may not be present in NER. These studies suggest that combined morphological and molecular methods should be employed for the correct identification of Anopheles species in NER. Morphological misidentification of NER vector as mainland vector can mislead vector control policies, causing public health havoc.