The conserved chromosome complement of culicid mosquitoes has long been recognized. Across the family Culicidae, most species share a diploid number of six chromosomes, typically organized as two pairs of autosomes and one pair of sex chromosomes. This remarkable stability contrasts sharply with many other insect groups, where chromosome fusions, fissions, and rearrangements frequently reshape karyotypes over evolutionary time. The Brazilian team set out to ask what this chromosomal conservatism actually conceals. If the number and gross morphology of chromosomes are so similar, does that mean the genomes themselves are similar? The answer, according to the new data, is an emphatic no. The study reveals that genome size, heterochromatin distribution, ribosomal DNA organization, and microsatellite landscapes can all diverge substantially even when the karyotype appears identical under the microscope.

To build this picture, the researchers applied an unusually broad toolkit to mitotic chromosomes prepared from the three species. Giemsa staining provided the baseline description of chromosome number and shape. C-banding then highlighted the distribution of constitutive heterochromatin, the densely packed, repeat-rich chromatin that tends to cluster around centromeres. Sequential staining with the fluorochromes chromomycin A3 and DAPI distinguished GC-rich from AT-rich regions, revealing compositional differences that ordinary stains cannot resolve. Fluorescence in situ hybridization, or FISH, allowed the team to physically map ribosomal DNA clusters and repetitive microsatellite motifs onto specific chromosomes. Finally, flow cytometry delivered precise measurements of nuclear DNA content, using the stingless bee Scaptotrigona xanthotricha as an internal reference standard. This integrative design meant that every claim about genome organization could be cross-checked against an independent measurement technique.

The flow cytometry results delivered the study’s headline surprise. The haploid genome size of Toxorhynchites violaceus was estimated at 1.199 picograms, while its congener Toxorhynchites theobaldi measured 1.003 picograms, and Lutzia bigoti came in at just 0.684 picograms. In relative terms, the genome of T. violaceus is roughly 75 percent larger than that of L. bigoti, a difference that would be expected to leave some visible trace in chromosome dimensions. Yet when the team subjected total chromosome length to statistical analysis, no significant differences emerged among the three species. This decoupling of genome size from chromosome size and number is the central conceptual contribution of the paper. It indicates that the extra DNA in the larger genomes is not organized into new chromosomes or visibly elongated arms, but is instead packed into regions of the genome that standard cytological preparation does not resolve as length differences.

What kind of DNA accounts for this hidden variation? The cytogenetic evidence points squarely at repetitive sequences, the so-called repeatome. C-banding showed that heterochromatin is consistently concentrated in pericentromeric regions in all three species, a pattern typical of many animal genomes. However, the amount and fine distribution of this heterochromatin varied between species, and the fluorochrome staining revealed species-specific patterns of GC-rich sequence accumulation. In both Toxorhynchites species, the team also documented heteromorphisms, cases where one homologous chromosome carried a larger CMA3-positive region or a ribosomal DNA site that its partner lacked. Such within-individual variation is a classic signature of recent transposition and unequal crossing over among tandem repeats, processes that can rapidly inflate or deflate specific genomic compartments without altering chromosome structure in any obvious way.

The ribosomal DNA findings added another layer of intrigue. In both Toxorhynchites species, FISH with an 18S rDNA probe labeled distinct chromosomal sites, and the overall organization of these ribosomal clusters appeared conserved between the two relatives. In Lutzia bigoti, however, no detectable rDNA signal was observed under the hybridization conditions used. The authors suggest this absence may reflect divergence in the structure of the ribosomal DNA repeats themselves, such that the probe no longer binds strongly enough to produce a visible signal. Rather than indicating that L. bigoti lacks ribosomal genes entirely, which would be biologically implausible, the result most likely points to sequence divergence or chromosomal relocation of the ribosomal arrays in this lineage. It is a reminder that even the most essential and conserved gene families in eukaryotic genomes can evolve lineage-specific quirks in their physical organization.

One of the most technically impressive aspects of the study involved chromosome microdissection. Using micromanipulation, the researchers isolated an individual chromosome and amplified its DNA to generate a chromosome-specific probe. When this probe was hybridized back onto chromosome spreads from both Toxorhynchites species, it painted the pericentromeric regions in each, confirming that repetitive DNA concentrated around the centromeres is at least partially shared between the two species. Intriguingly, however, the labeled chromosome pair differed between T. theobaldi and T. violaceus, meaning that the same repeat family occupies different chromosomal addresses in the two genomes. Combined with the species-specific microsatellite distribution patterns documented by FISH, this result paints a picture of a repeatome in constant flux, with sequences being amplified, eroded, and redeployed across the karyotype even between closely related species.

The broader significance of the work lies in what it says about genome evolution in the Culicidae as a whole. Mosquito genome sizes reported across the family span a considerable range, and previous studies have linked this variation primarily to the content of transposable elements and other repeats rather than to whole-genome duplications. The new data from T. theobaldi, T. violaceus, and L. bigoti reinforce that interpretation with an unusually clean example: three species with indistinguishable karyotypes but genome sizes differing by up to three-quarters. The authors conclude that genome evolution in mosquitoes is driven primarily by changes in the content and organization of repetitive DNA rather than by alterations in chromosome number or large-scale morphology. In other words, the karyotype is a poor proxy for genomic diversity, and studies that rely on chromosome counts alone will systematically underestimate the evolutionary dynamism of these insects.

There are also practical implications for research on mosquito biology and control. Toxorhynchites larvae are predatory on other mosquito larvae, and several species in the genus have been explored as biological control agents against disease vectors. Lutzia bigoti, likewise, is a natural predator of container-breeding mosquitoes in the Neotropics. Understanding the genome organization of these non-hematophagous relatives not only fills a gap in comparative cytogenetics but may also inform future genomic and transgenic approaches to vector control, where knowledge of repeat landscapes matters for genome assembly, transgene insertion, and the design of gene drive systems. The finding that rDNA organization can diverge to the point of probe non-detection between genera underscores how much basic cytogenetic ground remains to be covered even within a single well-known insect family.

The study, led by Mara Garcia Tavares and Wellington Ronildo Clarindo with colleagues in the Department of General Biology at the Universidade Federal de Viçosa, was published open access in Parasites & Vectors, with the accepted manuscript released ahead of the final version of record. The team notes that its integrative approach, pairing flow cytometric genome sizing with multi-probe FISH and microdissection-derived probes, offers a template for future work on understudied taxa. As sequencing costs continue to fall, cytogenetic ground-truthing of the kind presented here will remain essential for interpreting assembled genomes correctly, particularly in repeat-rich regions where short-read assembly struggles. For now, the message from these three Neotropical mosquitoes is clear: beneath a calm and unchanging chromosomal surface, their genomes are churning with repetitive DNA, and that hidden churn is the real engine of mosquito genome evolution.

Subject of Research: Comparative cytogenetics and genome size evolution in Neotropical mosquitoes

Article Title: Integrative cytogenetic and genome size analyses reveal conserved karyotypes and repetitive DNA-driven genome variation in the mosquitoes Toxorhynchites theobaldi, Toxorhynchites violaceus, and Lutzia bigoti

Article References: Tavares, M. G., Lopes, A. L. G., Soares, A. L. M., Soares, F. A. F., Martins, G. F., & Clarindo, W. R. (2026). Integrative cytogenetic and genome size analyses reveal conserved karyotypes and repetitive DNA-driven genome variation in the mosquitoes Toxorhynchites theobaldi, Toxorhynchites violaceus, and Lutzia bigoti. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07641-5

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07641-5

Keywords: Culicidae, karyotype, repetitive DNA, genome size, flow cytometry, fluorescence in situ hybridization, rDNA, microsatellites, heterochromatin, chromosome microdissection, Toxorhynchites, Lutzia bigoti

The research team, led by Diana G. Argaez-Sierra and colleagues at the Universidad Autónoma de Yucatán’s Arbovirology Laboratory, set out to inventory the mosquito fauna of a reserve that had received surprisingly little focused entomological attention despite its ecological prominence. The Ría Celestún Biosphere Reserve is one of the largest of Yucatán’s twelve protected natural areas, encompassing roughly 81,482 hectares of mangroves, coastal dune vegetation, salt marshes, petenes, and lowland floodplain forest on the Gulf of Mexico. The region’s warm, semi-arid climate brings about 767 millimeters of annual rainfall, with relative humidity ranging from 65 to 95 percent and maximum monthly temperatures climbing to 40 degrees Celsius—a combination that creates ideal conditions for mosquito breeding across a mosaic of brackish and freshwater habitats.

Sampling took place during the rainy season in October and November 2024, when the researchers deployed a cross-sectional design across 27 sites representing seven distinct habitat types, including mangrove swamp, salt flats, water surge zones, the bird-nesting islet known as Isla de los Pájaros, coastal scrub, disturbed vegetation, and coastal halophytic vegetation. Working from 19:00 to 22:00 and again from 05:00 to 08:00, the team used two backpack-mounted Prokopack aspirators per night to vacuum adult mosquitoes directly from shaded areas and dense vegetation. They supplemented this active collection with BG-Sentinel traps baited with synthetic human-scent attractant, five traps per site running overnight. Captured insects were immediately preserved in liquid nitrogen and transported to the laboratory, where stereomicroscopes and standard taxonomic keys were used to identify species morphologically.

The results were dominated by a single, familiar face. Of the 3,755 adult mosquitoes captured, females of Aedes taeniorhynchus—the black salt-marsh mosquito—accounted for 95.26 percent of the entire collection and turned up in every habitat sampled. This species is famously associated with coastal wetlands and mangroves throughout the Americas, ovipositing in brackish-water environments and displaying a remarkable tolerance for salinity that few other mosquitoes can match. Its larvae can even colonize artificial containers, allowing the insect to bridge wild and human-modified landscapes. While Ae. taeniorhynchus is primarily a biting nuisance in much of its range, its epidemiological resume is sobering: Venezuelan equine encephalitis virus has been isolated from this species in Guatemala and Costa Rica, eastern equine encephalitis virus in South Carolina, and the Itaqui virus, which causes fever and headache in humans, in Venezuela.

The headline discovery of the survey, however, was Anopheles gabaldoni, a mosquito first described in Tabasco, Mexico in 1941 and subsequently reported in Veracruz and Chiapas, as well as in the neighboring Yucatán Peninsula states of Campeche and Quintana Roo. Its capture in the disturbed vegetation area and on Isla de los Pájaros marks the first record for Yucatán state, raising the state’s documented mosquito tally to 60 species. The identification rests on distinctive abdominal morphology—tufts of posterolateral scales on the tergites and numerous light and dark scales on the sternites—features that the team documented photographically. Intriguingly, all Anopheles species in the study except An. vestitipennis were collected on the islet, including An. albimanus, An. crucians, An. punctipennis, and An. pseudopunctipennis, suggesting that this mangrove-covered bird sanctuary functions as an anopheline hotspot within the reserve.

Habitat type proved to be a strong predictor of mosquito diversity. The disturbed vegetation area yielded the richest assemblage with 12 species, followed by Isla de los Pájaros and the floodable area with seven species each. Although forest modification is generally associated with reduced species diversity, the authors propose that the convergence of disturbed vegetation and highly conserved mangrove may actually amplify mosquito richness by creating edge habitats that satisfy the breeding requirements of multiple species simultaneously. Six of the 27 sampling sites produced no mosquitoes at all, including two sites in the saltworks and three on the islet, underscoring how patchy mosquito distributions can be even within favorable landscapes.

Because morphology alone can mislead—Coquillettidia venezuelensis resembles Cq. nigricans, Anopheles vestitipennis resembles An. neivai, and species of the Aedes subgenus Ochlerotatus are notoriously similar—the team turned to DNA barcoding to confirm the identities of three species. Using the Folmer primers LCO1490 and HC02198, they amplified roughly 700-base-pair fragments of the mitochondrial cytochrome c oxidase subunit I gene. The 704-base-pair COI sequence of An. vestitipennis showed 99.56 percent nucleotide identity with reference sequences in GenBank, and the 687-base-pair sequence of Cq. venezuelensis reached 99.85 percent identity. For Ae. angustivittatus, the 707-base-pair COI sequence showed 98.50 percent identity with a specimen from neighboring Quintana Roo, and a complementary 377-base-pair fragment of the nuclear ITS2 region was also generated. All sequences were deposited in GenBank under accession numbers PZ154394, PZ154395, PZ154396, and PZ158059, adding a permanent molecular reference resource for future mosquito work in the region.

The phylogenetic analysis added a tantalizing evolutionary twist. Bayesian inference conducted in MrBayes, using a 46-sequence dataset of 579 nucleotide positions and Culex quinquefasciatus as an outgroup, revealed that the Celestún Ae. angustivittatus forms a strongly supported lineage with the Quintana Roo specimen, with 98 percent posterior support. But when the researchers calculated pairwise genetic distances, the Yucatán sequences diverged by 3.11 to 4.15 percent from Ae. angustivittatus collected in Chiapas, the State of Mexico, Colombia, and Ecuador. Divergence values in the 3 percent range are often taken as evidence of distinct evolutionary lineages in mosquito DNA barcoding, and the authors suggest that what is currently called Ae. angustivittatus may actually comprise multiple species or distinct subpopulations across its range. They ruled out confusion with the closely related Aedes infirmatus, since interspecific divergence between the two species measured only 2.76 to 2.77 percent, and they note that the ITS2 marker was of limited use here because public databases contain too few ITS2 sequences from closely related Aedes species. Resolving the question will require broader sampling, diagnostic morphological comparisons, and potentially complete mitochondrial genome analysis.

The public health stakes of the inventory are considerable. The Ría Celestún Biosphere Reserve has previously yielded Cache Valley and South River viruses and alphavirus RNA from Ae. taeniorhynchus, alphavirus RNA from An. pseudopunctipennis and An. crucians, and St. Louis encephalitis virus isolated from the bird Cyanocorax yncas. Females of Ae. taeniorhynchus captured at the Mérida zoo tested positive for Cache Valley and Kairi viruses, both of which primarily infect livestock and wildlife but can incidentally infect humans. In Colombian mangrove forests, RNA from St. Louis encephalitis, yellow fever, dengue, Venezuelan equine encephalitis, and West Nile viruses has been detected in mosquitoes, and Venezuelan equine encephalitis virus and St. Louis encephalitis virus have been found in Mansonia titillans—one of the eight species newly recorded at Celestún. With the reserve attracting heavy tourism and hosting dense communities of birds and mammals, the presence of competent vector species creates precisely the ecological conditions under which zoonotic arboviruses can circulate undetected until they spill over into human populations.

Methodologically, the study also delivered a practical lesson for surveillance design: the backpack aspirator captured 94.70 percent of all mosquitoes across 15 species, while the BG-Sentinel traps, which excel at collecting human-biting species like Ae. aegypti, accounted for just 5.30 percent across nine species. For rare species such as Ae. angustivittatus, An. vestitipennis, Cq. venezuelensis, and Ma. titillans, both methods contributed in similar proportions, suggesting that combining techniques remains essential for a complete picture of diversity. The authors are candid about the limitations of a single wet-season pass through the reserve; each site was inspected only once, and dry-season sampling will be needed to capture seasonal shifts in species composition. They also call for intensified arbovirus testing on Isla de los Pájaros, whose hundreds of nesting pelicans, herons, frigatebirds, and cormorants make it a natural laboratory for understanding how mosquitoes, birds, and viruses interact in one of the Yucatán’s most ecologically vital corners. With 24 species now documented across the reserve—40 percent of Yucatán state’s entire mosquito fauna—Celestún has firmly established itself as a frontier for entomological discovery on Mexico’s Gulf coast.

Subject of Research: Mosquito species inventory and DNA barcoding in a Mexican mangrove biosphere reserve

Article Title: Mosquito fauna (Diptera: Culicidae) in the Ria Celestún Biosphere Reserve (RCBR) with a new record for Yucatán, México

Article References: Argaez-Sierra, D. G., Cigarroa-Toledo, N., Baak-Baak, C. M., Tzuc-Dzul, J. C., Cetina-Trejo, R. C., Talavera-Aguilar, L. G., Chi-Chim, W. A., Arana-Guardia, R., & García-Rejón, J. E. (2026). Mosquito fauna (Diptera: Culicidae) in the Ria Celestún Biosphere Reserve (RCBR) with a new record for Yucatán, México. Discover Animals, 3(1), Article 93. https://doi.org/10.1007/s44338-026-00253-4

Image Credits: AI Generated

DOI: 10.1007/s44338-026-00253-4

Keywords: mosquitoes, Culicidae, Anopheles gabaldoni, Ría Celestún Biosphere Reserve, Yucatán, DNA barcoding, COI gene, arboviruses, Aedes taeniorhynchus, mangroves, biodiversity, vector surveillance

The research team, led by Cecília Ferreira de Mello and Jeronimo Alencar of the Oswaldo Cruz Foundation, worked at Terra Boa Farm in the municipality of Silva Jardim, a roughly three-hectare agroecological plot where açaí palms and peach palms are cultivated alongside native trees. Between June 2022 and December 2025, the team carried out 43 monthly sampling events totaling 86 hours of collection effort. Rather than focusing on a single habitat type, as most previous studies have done, they systematically sampled five distinct larval environments: three natural ones, namely bromeliads, bamboo internodes, and fallen peach palm leaf bracts, and two artificial ones, a plastic tarp laid on the ground where rainwater pooled in its depressions, and a 200-liter polyethylene water barrel. None of these habitats were introduced by the researchers; they were all features already present in the working farm.

In total, the team collected 5,314 immature mosquitoes. The genus Culex dominated numerically, accounting for 53.8 percent of all specimens, followed by Limatus at 16.6 percent and Wyeomyia at 16.2 percent. Just five species, Culex iridescens, Culex pleuristriatus, Limatus durhamii, Wyeomyia edwardsi, and the Asian tiger mosquito Aedes albopictus, together made up nearly 80 percent of everything captured. The statistical analysis, which included individual-based species accumulation curves computed with rarefaction and extrapolation methods, bipartite interaction networks linking each species to each habitat, and cluster analysis based on Jaccard dissimilarity, revealed a community structured far more tightly than chance alone would predict. Each habitat type hosted a recognizable assemblage of specialists, a pattern ecologists describe as niche partitioning.

The natural habitats overwhelmingly won the diversity contest. Bromeliads, bamboo internodes, and peach palm bracts together yielded 4,175 specimens, or 78.6 percent of the total, and supported 34 of the 38 recorded species. Twenty-six species occurred exclusively in natural habitats. Bromeliads alone produced 2,031 individuals from 22 species, with the specialists Cx. pleuristriatus and Wy. edwardsi making up more than four-fifths of the bromeliad catch. Bamboo internodes, dominated by Cx. iridescens at 72.4 percent of their fauna, supported 21 species and even harbored predatory larvae of Toxorhynchites trichopygus, a genus whose adults do not bite and whose larvae may help regulate populations of other mosquitoes by preying on them. The species accumulation curves for natural habitats did not reach an asymptote, hinting that the true richness of these phytotelmata, particularly in the unsampled forest canopy, is likely even higher.

The artificial habitats told a very different story. The plastic tarp and the water barrel together accounted for 1,139 specimens but only 12 species, just four of which were exclusive to artificial containers. The water barrel was nearly barren, yielding a mere 22 individuals of six species, with Aedes albopictus making up almost two-thirds of them. The tarp, by contrast, was a productive breeding ground, hosting 1,117 specimens from nine species, dominated by Li. durhamii and Ae. albopictus, which together represented 83.7 percent of its mosquito fauna. Perhaps most striking was the consistent presence of Culex quinquefasciatus, the southern house mosquito, a species typically confined to urban and heavily degraded environments, breeding in plastic tarps embedded within a farm surrounded by preserved forest. The authors interpret this as evidence of increased ecological plasticity under agroforestry management conditions.

The epidemiological implications are where the study becomes genuinely alarming for public health. Among the species recorded were Haemagogus leucocelaenus and Haemagogus janthinomys, the primary vectors of sylvatic yellow fever virus in the Americas, colonizing bamboo stands within the farm. The team also documented Sabethes identicus, a rarely recorded species considered a secondary yellow fever vector. Brazil experienced a major yellow fever outbreak between 2016 and 2018 driven by these very mosquito genera, and the presence of their larvae in an agricultural landscape adjacent to protected areas such as the Poço das Antas ecological reserve suggests that agroforests could serve as staging grounds where sylvatic vectors and farmworkers come into close contact.

Then there is Aedes albopictus, the Asian tiger mosquito, which emerged as the study’s great generalist. It was the only species detected in all five habitat types, thriving equally in natural bromeliads and bamboo, in ephemeral peach palm bracts, and in human-made tarps and barrels. This versatility matters because Ae. albopictus is a recognized bridge vector, capable of moving pathogens between sylvatic transmission cycles in wildlife and urban cycles in human populations. Recent laboratory work has demonstrated its competence for yellow fever virus, raising concerns about possible reemergence of urban yellow fever in Brazil. A mosquito that breeds in both the forest and the farmyard, and that readily bites humans, represents precisely the kind of conduit through which arbovirus spillover could occur.

The cluster analysis of species composition added a layer of ecological nuance. Bromeliads and bamboo internodes, both stable, long-lasting natural phytotelmata, hosted the most similar communities, as did peach palm bracts and the plastic tarp, both of which are relatively ephemeral, sun-exposed pools. The water barrel stood apart from everything else, hosting the most distinct assemblage. This pattern indicates that oviposition site selection by gravid female mosquitoes is far from random: it is governed by water chemistry, shading, container geometry, and chemical cues, with each habitat type acting as an environmental filter that sorts the regional species pool into distinct local communities.

Importantly, the authors are not arguing against agroforestry. These systems are widely promoted as sustainable alternatives to deforestation and monoculture, reconciling agricultural production with biodiversity conservation, and the sheer diversity of mosquitoes recorded at Terra Boa Farm, including many specialized, non-vector species, underscores their ecological value. Instead, the researchers propose practical management measures that farmers can adopt without destroying the natural habitats that sustain predators and specialists. Unused plastic tarps should be inspected, rolled up, or properly disposed of after rainfall to eliminate breeding sites for Ae. albopictus and Cx. quinquefasciatus. Water storage barrels should be tightly sealed or fitted with fine mesh screens. Meanwhile, bromeliads and bamboo internodes should be left intact, because they support natural enemies such as Toxorhynchites larvae and specialized non-vector species that contribute to ecosystem stability and may competitively displace vector taxa.

The study also lays out a clear agenda for future research. Because the current work assessed larval communities rather than adult infection status, the authors call for molecular screening of mosquitoes for arboviruses and longitudinal studies of vector-host contact at the forest-agriculture interface. Such work would move beyond theoretical assessments of vector potential and allow direct measurement of pathogen spillover risk. As agroforestry expands across the Atlantic Forest as a policy tool for ecological restoration and income generation, understanding these landscapes as eco-epidemiological interfaces, where sylvatic pathogen cycles, vectors, domestic animals, and agricultural workers converge, will be essential for balancing the conservation benefits of sustainable farming against the public health risks that come with bringing forest and humanity closer together.

Subject of Research: Mosquito community structure in natural and artificial larval habitats within an Atlantic Forest agroforestry system in Rio de Janeiro, Brazil

Article Title: Phytotelm and anthropogenic mosquitoes: community structure in natural and artificial habitats in an Atlantic Forest agroforestry system, Rio de Janeiro, Brazil

Article References: de Mello, C. F., Müller, G. A., de Alcantara Azevedo, W. T., Gil-Santana, H. R., Araújo, T. R., Pimentel, R., & Alencar, J. (2026). Phytotelm and anthropogenic mosquitoes: community structure in natural and artificial habitats in an Atlantic Forest agroforestry system, Rio de Janeiro, Brazil. The Science of Nature, 113(5), Article 113. https://doi.org/10.1007/s00114-026-02162-8

Image Credits: AI Generated

DOI: 10.1007/s00114-026-02162-8

Keywords: mosquitoes, Culicidae, phytotelmata, agroforestry, Atlantic Forest, Aedes albopictus, Haemagogus, yellow fever, arbovirus spillover, larval habitats, bromeliads, vector surveillance