The study comes at a critical moment for global dengue control. Dengue is the most rapidly expanding arboviral infection worldwide, with cases reported to the World Health Organization escalating from 505,430 in 2000 to 14.6 million in 2024. Once endemic in only nine countries in 1970, the disease has now spread to more than 120 countries, and India alone recorded 121,824 confirmed dengue cases and 131 deaths in 2025. With no universally efficacious vaccines or therapeutics available, vector control remains the principal strategy against the disease, yet the widespread emergence of insecticide resistance in Ae. aegypti populations has significantly compromised conventional insecticide-based approaches, necessitating the urgent exploration of alternatives such as the Sterile Insect Technique and Wolbachia-based vector control.

Wolbachia is a genus of Gram-negative, intracellular, maternally transmitted endosymbiotic bacteria first discovered as Rickettsia-like organisms from the germ cells of Culex pipiens mosquitoes. Natural Wolbachia infection has been reported in more than 60 percent of all insect species, but Ae. aegypti, the major vector of dengue, is generally considered free of natural Wolbachia infection, which is why researchers have transinfected laboratory strains with the wMel and wAlbB strains. When present in mosquitoes, Wolbachia can manipulate host reproductive biology through cytoplasmic incompatibility, in which embryos fail to develop when Wolbachia-free females mate with Wolbachia-infected males. This mechanism underpins both population replacement strategies, which involve releasing infected males and females to gradually substitute the natural mosquito population, and population suppression approaches, which involve releasing only infected males to produce non-viable offspring.

Field release trials of Wolbachia-infected mosquitoes conducted in Australia, Indonesia, Brazil, Colombia, and Malaysia have shown significant reductions in notified dengue and chikungunya cases. The trial in Yogyakarta, Indonesia, is regarded as the gold standard, resulting in a 77 percent reduction in virologically confirmed dengue incidence in treated areas compared with untreated areas over 27 months. Currently, field releases of wMel-infected Ae. aegypti are underway in 11 countries. A joint venture between ICMR-NIVCR and the World Mosquito Program from Monash University developed two unique Indian Ae. aegypti strains carrying wMel and wAlbB through multiple backcrosses between Wolbachia-infected Australian females and uninfected wild Puducherry males over six generations. The operationalization of Wolbachia-based vector control in field conditions requires large-scale rearing and release of mosquitoes with optimum fitness characteristics, making larval diet a pivotal factor in mass mosquito rearing.

The new study evaluated the effects of four commercially available larval diets on fitness characteristics of Wolbachia-transinfected and uninfected Ae. aegypti strains. The diets tested were LD1, a fish feed known as TetraBits granules; LD2, a laboratory rodent diet; LD3, a mushroom powder derived from 100 percent natural button mushroom Agaricus bisporus; and LD4, a mix of dog biscuit and brewer’s yeast in a 3:2 ratio. Dog biscuits provide proteins, animal fat, calcium, and vitamins, while brewer’s yeast is rich in proteins, starch, sugars, and minerals. Mushroom powder offers a plant-based nutritional source containing proteins, carbohydrates, vitamins, minerals, dietary fibers, and various bioactive compounds. Each diet was provided at approximately 0.06 grams of food per larva on alternate days, with all experiments initiated simultaneously under controlled conditions of 27 plus or minus 2 degrees Celsius, 80 plus or minus 10 percent relative humidity, and a 12-hour photoperiod.

The experimental design comprised four larval diet regimens, three mosquito strains, and two generations, yielding a full factorial design of 24 diet-strain-generation combinations. Each combination was assessed using three independent biological replicates, with each replicate initiated from 100 first-instar larvae, for a total of 7,200 larvae across the full design. Wing length was measured from the alular notch to the wing tip using a stereomicroscope fitted with a digital camera, and body weight was measured individually using a semi-micro analytical balance with a readability of 0.01 milligrams after drying samples at 60 degrees Celsius for 12 hours. Wolbachia density was measured using duplex real-time PCR targeting either the Wolbachia surface protein gene specific to wMel or the ankyrin repeat domain specific to wAlbB, with the ribosomal protein 17 gene of Ae. aegypti as the reference. Relative density was determined using the delta Ct method.

The results revealed complex interactions among diet, Wolbachia strain, and generation. Female wing length was significantly influenced by the three-way interaction between diet, strain, and generation, with an F statistic of 3.98 and a P value of 0.0044, together with a significant Diet by Strain interaction. In contrast, male wing length was primarily affected by diet alone, with an F statistic of 14.11 and P less than 0.001, and by the Diet by Generation interaction. Male body weight also showed a significant three-way interaction among diet, strain, and generation, indicating strain- and generation-specific dietary responses. Female body weight exhibited significant Diet by Strain and Strain by Generation interactions. Overall, larval diet was the primary determinant of mosquito fitness, with its effects modified by Wolbachia infection status and generation. Female wing length was consistently greater than male wing length across all diet groups and strains, and across most dietary treatments, F1 mosquitoes tended to show slightly greater wing lengths than F0 mosquitoes.

LD1 and LD4 showed improved wing development and fitness compared to LD2 and LD3, with sex-specific effects observed. In the F0 generation, the LD2 diet yielded the highest mean female wing length, although it did not differ significantly from LD1 or LD4, and all three diets produced significantly larger wing lengths than LD3. In the F1 generation, the LD4 diet resulted in the highest mean female wing length and was significantly superior to LD3. Male wing length ranged between 2099 and 2271 micrometers, with LD3 consistently producing the shortest wing lengths across all strains. The wMel strain on LD2 diet showed the lowest female wing length at 2657.03 micrometers. Notably, the LD2 diet in the F1 generation of the wMel strain could not be completed, as no adults survived to a stage permitting assessment of wing length or body weight, a finding consistent with previous reports where nutritionally imbalanced or single-source larval diets resulted in complete failure to pupate, suggesting toxicity or critical nutrient deficiency.

Wolbachia density measurements revealed substantial variation across larval diets, generations, and mosquito strains. The wAlbB strain exhibited higher Wolbachia densities than the wMel strain across most diet treatments. Higher densities were generally observed in mosquitoes reared on fish feed and laboratory rodent diet, whereas mosquitoes reared on the dog biscuit and brewer’s yeast diet exhibited comparatively lower Wolbachia densities. Within the wAlbB strain, LD2 produced no significant change in density compared to LD1, while LD4 led to a significant reduction with a beta coefficient of minus 10.38 and P less than 0.001. The wMel strain generally exhibited lower Wolbachia density than wAlbB, and although LD4 also reduced density in wMel, this reduction was less pronounced compared to wAlbB, with a significant positive interaction between LD4 and wMel. The mushroom powder treatment did not show any detectable Wolbachia density in any of the replicates and was excluded from the analysis.

These findings have direct implications for Wolbachia-based mass-rearing and release programmes, where standardized larval diets are often applied uniformly across strains. The results suggest that diet formulations may need to be strain-specific rather than generalized, to ensure optimal larval development, adult body size, and Wolbachia density are simultaneously achieved. Wolbachia directly competes with its mosquito host for amino acids, and the lack of independent fatty acid and cholesterol synthesis pathways in Wolbachia drives further reliance on host-derived lipid resources. Such resource competition could impose an additional metabolic burden on Wolbachia-infected strains, particularly under suboptimal dietary conditions, potentially explaining the diet-dependent divergence in body weight observed between strains. Variations in Wolbachia density can influence key phenotypic traits including cytoplasmic incompatibility, pathogen blocking, and maternal transmission, thereby affecting the effectiveness and sustainability of Wolbachia dissemination in target mosquito populations. Based on the analysis, LD1 is recommended for routine mass rearing of Wolbachia-transinfected mosquitoes, and together with LD4, demonstrated superior performance for both Wolbachia-free and transinfected strains under laboratory conditions. The findings highlight the need for future studies on cost-effective, locally available diet formulations to optimize mosquito fitness for vector control strategies, and suggest investigations into the complex interactions between dietary components, native microbiota, and Wolbachia density patterns to optimize both mosquito fitness and symbiont stability for successful population replacement and suppression strategies.

Subject of Research: Larval diet-mediated morphological trait and Wolbachia density variation in Wolbachia-transinfected (wMel and wAlbB) and Wolbachia-free Aedes aegypti mosquitoes

Article Title: Larval diet-mediated morphological trait and Wolbachia density variation in Wolbachia-transinfected (wMel and wAlbB) and Wolbachia-free Aedes aegypti mosquitoes

Article References: Revi, A., Gunasekaran, Y., Pachalil Thiruvoth, V., Ramasamy, A., Arif, W., Kuppusamy, M., Venugopal, D., Ebinezar, K., Bakianathan, E., Essoudasse, A., Parandamane, P., Vijayan, A., Anbazhagan, T., Nirmala Mary, E., Ramkumar, H., Shriram, A. N., & Rahi, M. (2026). Larval diet-mediated morphological trait and Wolbachia density variation in Wolbachia-transinfected (wMel and wAlbB) and Wolbachia-free Aedes aegypti mosquitoes. Frontiers in Insect Science, 6, Article 1887426. https://doi.org/10.3389/finsc.2026.1887426

Image Credits: AI Generated

DOI: 10.3389/finsc.2026.1887426

Keywords: Aedes aegypti, Wolbachia, wMel, wAlbB, larval diet, dengue, vector control, mosquito fitness, Wolbachia density, mass rearing, Larval, diet-mediated