Genetic Variability and Population Structure within the Anopheles tessellatus complex (Theobald, 1901) in Indonesia using ITS2 nuclear and COI, COII mitochondrial sequences

This study utilizes ITS2, COI, and COII molecular markers to reveal that the *Anopheles tessellatus* complex in Indonesia comprises three distinct genetic lineages across five islands with high haplotype diversity and potential speciation in specific populations, highlighting the need for further research to clarify its role in malaria transmission.

The Gist

Imagine you have a box of 1,000 identical-looking white t-shirts. To the naked eye, they all look exactly the same. You might assume they are all made in the same factory, by the same people, and are all the same size.

But what if I told you that hidden inside the fabric of these shirts are tiny, invisible "stitch marks" that prove they were actually made in three different factories, by three different groups of people, who haven't spoken to each other in thousands of years?

That is essentially what this scientific paper is about, but instead of t-shirts, the researchers are studying mosquitoes.

The Mystery of the "Look-Alike" Mosquito

The mosquito in question is called Anopheles tessellatus. In Indonesia, this mosquito is a suspected "suspect" in spreading malaria and lymphatic filariasis (a disease that causes severe swelling).

For a long time, scientists thought all these mosquitoes were just one big family. They looked the same under a microscope, so they were treated as one single species. But in the world of mosquitoes, looking alike doesn't always mean being related. Just like identical twins can have very different personalities, these mosquitoes might look the same but have very different behaviors (like where they bite or how they spread disease).

The Genetic "DNA Fingerprint" Hunt

To solve this mystery, the researchers didn't just look at the mosquitoes' wings or legs. Instead, they went into the mosquito's "library" (its DNA) and read three specific "chapters" of its genetic book:

1. ITS2: A nuclear gene (like the family recipe book).
2. COI & COII: Mitochondrial genes (like the mother's family heirloom jewelry).

Think of these genes as barcodes. Even if two mosquitoes look identical, their barcodes might be slightly different, revealing their true family history.

The Big Discovery: Three Secret Clans

When the researchers scanned the barcodes of mosquitoes collected from all over Indonesia (from Sumatra in the west to Papua in the east), they found something surprising. The "one big family" was actually three distinct clans that had been drifting apart for a long time:

• Clan Sumatra: The mosquitoes from the island of Sumatra had their own unique genetic signature.
• Clan Sulawesi: The mosquitoes from Sulawesi (an island shaped like a weird "K") were totally different from the others. They were so unique they seemed to be on their own evolutionary path.
• Clan Java & The Islands: The mosquitoes from Java, and the islands to the east (like Lombok and Sumba), formed a third group.

The Analogy: Imagine a group of friends who all wear the same uniform. You think they are all from the same school. But when you check their birth certificates (DNA), you realize one group is from a school in New York, one is from a school in London, and one is from a school in Tokyo. They look the same, but they have different histories and might behave differently.

Why Does This Matter?

This isn't just a fun fact for biology nerds; it's a huge deal for public health.

1. Different Personalities, Different Dangers: Just because they look the same doesn't mean they act the same. The "Sumatra Clan" might love to bite humans indoors at night, while the "Sulawesi Clan" might prefer biting cows outdoors. If the government tries to stop malaria by spraying indoor insecticides, they might stop the Sumatra clan but miss the Sulawesi clan entirely.
2. The "Speciation" Process: The study found that some of these groups (especially in Sulawesi and West Sumatra) are so genetically different that they might be in the process of becoming new species. They are like siblings who have moved out, started their own families, and are slowly becoming strangers.
3. High Diversity, Low Differences: The study found that while there are many different versions of these mosquitoes (high variety), the actual differences between them are small (low genetic distance). It's like having 50 different flavors of vanilla ice cream. They are all vanilla, but the subtle differences matter if you are a very picky eater (or a very picky mosquito).

The Takeaway

This paper is a wake-up call for Indonesia's health officials. You can't treat all Anopheles tessellatus mosquitoes as the same enemy.

• Old Way: "We see a mosquito. We spray everywhere."
• New Way: "We see a mosquito. Let's check its DNA barcode first. Is it from the Sumatra clan or the Sulawesi clan? Then we spray exactly where that specific clan lives."

By understanding these hidden genetic families, scientists can create smarter, more targeted plans to stop malaria and save lives, rather than just guessing. It's like realizing that to fix a leaky roof, you need to know exactly which shingles are broken, not just that the roof is wet.

Technical Summary

1. Problem Statement

• Taxonomic Complexity: Anopheles tessellatus is a recognized potential vector for malaria and lymphatic filariasis in South, East, and Southeast Asia. However, it belongs to a species complex where sibling species are morphologically indistinguishable (cryptic species).
• Control Challenges: Traditional morphological identification is insufficient to differentiate vectors from non-vectors within the complex, hindering effective malaria control strategies.
• Knowledge Gap: While An. tessellatus is prevalent in Indonesia, there is a lack of in-depth molecular studies regarding its genetic variation, population structure, and the existence of cryptic lineages across the diverse Indonesian archipelago.

2. Methodology

The study employed a multi-locus molecular approach to analyze genetic diversity and phylogenetic relationships.

• Sample Collection:
  • Scope: Adult mosquitoes were collected from 13 provinces across Indonesia (Sumatra, Java, Kalimantan, Lesser Sunda Islands, Sulawesi, and Papua) between 2015 and 2024.
  • Methods: Human landing catches, cattle-bait collections, CDC light traps, and animal-baited traps.
  • Specimens: 24 study samples were analyzed, supplemented by reference sequences from GenBank (Thailand, Malaysia, China, Philippines, etc.).
• Molecular Markers: Three genetic loci were targeted:
  1. Nuclear: Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA.
  2. Mitochondrial: Cytochrome c oxidase subunit I (COI) and subunit II (COII).
• Laboratory Procedures:
  • DNA extraction using Zymo Quick-DNA Miniprep Plus.
  • PCR amplification with specific primer sets for ITS2, COI, and COII.
  • Sanger sequencing and sequence editing (Sequencing Analysis v5.2).
• Bioinformatics & Statistical Analysis:
  • Alignment: MUSCLE v.5.3 and ClustalW.
  • Phylogenetics: Maximum Likelihood (ML) trees constructed using IQ-TREE v.2.3.6 with ModelFinder for model selection and UFBoot2 for support values.
  • Diversity Metrics: Haplotype diversity ($H_d$), nucleotide diversity ($\pi$), and pairwise genetic distances calculated using DnaSP v.6.12.03 and R packages (APE, SPIDER).
  • Network Analysis: Haplotype networks constructed using PopArt (TCS algorithm).

3. Key Results

A. Phylogenetic Structure and Lineages

The study identified that An. tessellatus in Indonesia forms a monophyletic group but is structured into three distinct genetic lineages (clusters) corresponding to geography:

1. Cluster 1 (Sumatra): Populations from West Sumatra.
2. Cluster 2 (Sulawesi): Populations from North and Central Sulawesi (Manado, Tojo Una-Una, North Morowali).
3. Cluster 3 (Java & Nusa Tenggara): Populations from Java, West Nusa Tenggara, East Nusa Tenggara, and parts of Sulawesi (Pangkajene Islands, Sigi, Palu).

• ITS2 Analysis: Revealed four distinct populations (Clades I–IV). Clade I was the most diverse and widespread in Indonesia. Clades III and IV showed specific geographic associations with Sulawesi and Sumatra/Kalimantan, respectively.
• COI Analysis: Identified 12 distinct lineages. While Lineage 1 was dominant and widespread, specific lineages were unique to West Sumatra, Garut (Java), and specific Sulawesi regions.
• COII Analysis: Confirmed the clustering pattern seen in COI, grouping samples into four clusters, with Cluster III (Sulawesi/Sumatra) showing higher intra-cluster divergence.

B. Genetic Diversity Metrics

• Haplotype Diversity ($H_d$):
  • COI: Highest diversity ($H_d = 0.914$), with 35 haplotypes identified.
  • COII: High diversity ($H_d = 0.931$), with 17 haplotypes.
  • ITS2: Moderate diversity ($H_d = 0.716$), with 5 haplotypes.
• Nucleotide Diversity: Generally low across all markers, suggesting recent demographic expansion.
• Genetic Distances:
  • Intraspecific distances were generally low (<1%).
  • However, specific populations (West Sumatra, Manado, Tojo Una-Una, North Morowali) showed genetic distances of 0.61–0.94% (COI), 0.81–0.95% (ITS2), and 0.62–0.71% (COII) relative to others, indicating potential incipient speciation.

C. Haplotype Networks

• Networks displayed a star-like topology for mitochondrial markers (COI and COII), with a central dominant haplotype (Hap2) connected to many singletons by short branches.
• This pattern suggests a recent population expansion following a historical bottleneck, likely driven by Pleistocene climatic fluctuations and sea-level changes in the Indonesian archipelago.
• Longer branches connected Indonesian haplotypes to those from Thailand, China, and Japan, indicating historical isolation or vicariance.

4. Key Contributions

1. First Comprehensive Molecular Survey: This is the first study to provide a detailed molecular characterization of An. tessellatus across the entire Indonesian archipelago using a multi-locus approach.
2. Revelation of Cryptic Structure: The study confirms that An. tessellatus is not a single homogeneous population but a complex of genetically distinct lineages associated with specific biogeographic regions (Sumatra, Sulawesi, Java/Nusa Tenggara).
3. Marker Validation: It validates the utility of combining nuclear (ITS2) and mitochondrial (COI, COII) markers. While ITS2 provided broader clustering, COI and COII offered higher resolution for detecting fine-scale population structure and recent demographic events.
4. Biogeographic Insights: The genetic structuring aligns with Indonesia's complex geological history, particularly the isolation of Sulawesi (Wallacean barrier) and the fragmentation of Sundaland.

5. Significance and Implications

• Vector Control Strategy: The identification of distinct genetic lineages suggests that different populations may possess varying vectorial capacities, ecological preferences, and behaviors (e.g., biting times, host choice). Current control measures based on a single "species" concept may be ineffective if these cryptic lineages differ in their response to interventions.
• Disease Surveillance: The findings underscore the need to incorporate molecular identification into national malaria and lymphatic filariasis surveillance programs to accurately track vector populations.
• Insecticide Resistance: High haplotype diversity implies that resistance traits could spread rapidly through gene flow. Monitoring specific lineages (especially the divergent Sumatra and Sulawesi clusters) is crucial for early detection of resistance.
• Future Research: The study calls for integrative taxonomic work (combining genomics with ecology and vector competence assays) to determine if these lineages represent distinct species. It also recommends whole-genome sequencing for higher resolution in future studies.

Conclusion: Anopheles tessellatus in Indonesia is a genetically structured species complex undergoing intraspecific diversification. The presence of cryptic lineages with potential for speciation necessitates a shift from morphological to molecular-based vector management strategies to support Indonesia's goal of malaria elimination by 2030.