Culex quinquefasciatus larvae development arrested when fed on Neochloris aquatica

Abstract

Culex quinquefasciatus is a cosmopolitan species widely distributed in the tropical and subtropical areas of the world. Due to its long history of close association with humans, the transmission of arboviruses and parasites have an important role in veterinary and public health. Adult females feed mainly on birds although they can also feed on humans and other mammals. On the other hand, larvae are able to feed on a great diversity of microorganisms, including microalgae, present in natural or artificial breeding sites with a high organic load. These two particularities, mentioned above, are some of the reasons why this mosquito is so successful in the environment. In this work, we report the identification of a microalga found during field sampling in artificial breeding sites, in a group of discarded tires with accumulated rainwater. Surprisingly, only one of them had a bright green culture without mosquito larvae while the other surrounding tires contained a large number of mosquito larvae. We isolated and identified this microorganism as Neochloris aquatica, and it was evaluated as a potential biological control agent against Cx. quinquefasciatus. The oviposition site preference in the presence of the alga by gravid females, and the effects on larval development were analyzed. Additionally, microalga effect on Cx. quinquefasciatus wild type, naturally infected with the endosymbiotic bacterium Wolbachia (w+) and Wolbachia free (w-) laboratory lines was explored. According to our results, even though it is chosen by gravid females to lay their eggs, the microalga had a negative effect on the development of larvae from both populations. Additionally, when the larvae were fed with a culture of alga supplemented with balanced fish food used as control diet, they were not able to reverse its effect, and were unable to complete development until adulthood. Here, N. aquatica is described as a biological agent, and as a potential source of bioactive compounds for the control of mosquito populations important in veterinary and human health.

Fig 1. Microalga morphotypes and molecular identification.

(A) Dendrogram of ITS1-5.8S-ITS2 rRNA region including the native N. aquatica and the closest related sequences from other microalgae available in NCBI database. Green diamond indicates the position of the native microalga. Number at nodes represents the percentages of bootstrap resampling based on 500 replicates. (B) Microalga morphologies at different light conditions, (a) autospores at constant light condition; (b), (c) and (d) cells in division with successive cleavages of protoplast at transition from constant light condition to darkness, (e) autospores at photoperiod condition and (f) vegetative cell at transition from photoperiod condition to darkness, single pyrenoid is indicated with arrow. All images were taken at 100x magnification. Bars represent 5 μm.

Fig 2. Selection of the oviposition site by gravid females of Cx. quinquefasciatus naturally infected with Wolbachia (w⁺) and Wolbachia free (w^-) isolines in N. aquatica suspension or dechlorinated water.

Female oviposition site selection was analyzed with binomial test (N = 30, p value = 0.05). ** p value < 0.001 indicate significant difference in oviposition site selection by gravid females from w⁺ isoline.

Fig 3. Survival curves of neonate Cx. quinquefasciatus larvae reared on N. aquatica suspension or dechlorinated water plus control diet (fish food).

Kaplan-Meier survival curves per oviposition substrate; α = 0.05. (A) Cx. quinquefasciatus w^- and (B) Cx. quinquefasciatus w⁺.

Fig 4. Development of Cx. quinquefasciatus lines fed on N. aquatica suspension (MS), microalga suspension plus control diet (MS+CD) and dechlorinated water with control diet (CD).

Proportion of larval stages, pupa and adult among diet treatments for Cx. quinquefasciatus w⁺ and w^- at different times. (A and D) at 5 days, (B and E) at 10 days, (C) at 15 days and (F) at 20 days. Graphs represent mean ± SEM of 3 independent experiments of 5 replicates each, initial n = 50 per experiment, statistical differences between development stages among diets were determined using 2-Way ANOVA (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

Fig 5. Cx. quinquefasciatus w^- reared on microalga suspension plus control diet.

Some adults were unable to emerge from pupae. Bar indicates 5 mm.

Fig 6. Total larvae length.

Cx. quinquefasciatus w^- and w⁺ larvae fed on control diet (CD), microalga suspension (MS) or microalga suspension plus control diet (MS+CD) at days 3, 7 and 11. Bars indicate 1 mm.

Fig 7. Effect of variables day, diet and isoline on total larvae length.

Larval lengths were measured at days 3 and 7, among diets: control diet (CD), microalga suspension (MS) and microalga suspension plus control diet (MS+CD) respectively; for both isolines (Isl) corresponding to w^- (solid color) and w⁺ (diagonal pattern).

Fig 8. Morphological alterations of Cx. quinquefasciatus w^- larvae fed on N. aquatica suspension under stereomicroscope observation.

Morphological alterations (MA) and midgut blockage (MB). (A) Larvae fed with control diet after 11 assay days, bar indicates 1mm. (B-F) Larvae fed with microalga suspension after 11 days. Bars indicate 0.5 mm.

Fig 9

(A) Larval midgut extracts of Cx. quinquefasciatus isolines w^- and w⁺ activity against N. aquatica cells. (a, c, e, g, i and k) bright field microscopy shows cells integrity, (b, d, f, h, j and l) fluorescence field microscopy shows chlorophyll autofluorescence. (a-b and g-h) microalga culture without midgut extract. Mixture of 10 μL of microalga plus 5 μL (c-d and i-j) or 10 μL of midguts extract (e-f and k-l). Bars represent 5 μm. Incubations were carried out at 37°C for 2 hours. (B) Different volumes of midgut extract (0.5, 1, 2, 5 and 10 μL) and microalga culture (10 μL) were mixed and incubated at 37°C for 2 hours or overnight, and then sown and incubated in BG-11 agar plates in controlled conditions of light and temperature, for 24 hours.