Mansonia are aggressive mosquito species that are abundant in aquatic ecosystems where the macrophyte plants occur. These mosquitoes are commonly found across the Amazon/Solimões River basin. However, little is known about the oviposition behavior of these species. In the present study, we registered observations on the oviposition activity of 3 species: Mansonia amazonensis, Ma. humeralis, and Ma. cf. titillans, in 5 species of macrophytes in the vicinities of the Madeira River, Porto Velho, State of Rondônia, Brazil. Overall, 197 egg batches were collected. A greater amount of egg batches was found in Salvinia molesta as compared with other macrophytes sampled. In addition, 2 new oviposition habitats were noted in Ludwigia helmintorrhiza and Limnobium spongia. These findings will be important to understand the reproductive dynamics of these mosquitoes in the Brazilian Amazon basin.
Mosquitoes of the genus Mansonia are widely found across the Brazilian Amazon River basin (Hutchings et al. 2008, Gama et al. 2012, Palermo et al. 2016, de Araújo et al. 2020, Hutchings et al. 2020). Six species were registered in the region: Mansonia amazonensis (Theobald), Ma. flaveola (Coquillett), Ma. humeralis Dyar and Knab, Ma. indubitans Dyar and Shannon, Ma. pseudotitillans (Theobald) and Ma. titillans (Walker) (Ferreira 1999, Hutchings et al. 2020, Galardo et al. 2022), all of which were recorded in the vicinities of the municipality of Porto Velho, State of Rondônia, Brazil (Galardo et al. 2022).
Aquatic ecosystems with macrophytes can be potential habitats for immatures of Mansonia species (Ronderos and Bachman 1963). The female leans on a substrate or water surface and bends the last abdominal segments to reach the abaxial part of the macrophyte leaf to lay numerous egg batches. The eggs are surrounded by a gelatinous substance, forming a cluster, organized in a rosette shape, a vision created due to the funneling of the apical pole of the eggs (Dyar and Knab 1916, Linley et al. 1986, Lounibos and Linley 1987). The main plant species associated with this underwater oviposition activity are the water hyacinth, Eichhornia crassipes (Mart.), and water lettuce, Pistia stratiotes L. (Dunn 1918, Laurence 1960). The immature forms have siphons (in the larvae) and trumpets (in the pupae) that are adapted to pierce the roots of these macrophytes to obtain oxygen from the aerenchyma of the host plants (Wesenberg-Lund 1918, Guille 1975). Observations in a colony of Mansonia (Mansonioides) revealed that first larval instars could not breathe for a long time without macrophytes, making this association essential for the survival of these mosquitoes (Samarawickrema 1968).
Although oviposition mechanisms of Mansonia are known, it is better documented for the subgenus Mansonioides, which has a restricted distribution in Africa and Asia (Laurence 1960). Water hyacinth and water lettuce are the most important macrophytes for oviposition and fixation of larvae and pupae (Lounibos and Linley 1987, Ferreira 1999) for the subgenus Mansonia (Nearctic and Neotropical distribution). However, little is known about the role of other macrophytes in the oviposition of these species in the Amazon/Solimões River basin.
Between February 24 and March 17 of 2022, we recorded, for the first time, batches of eggs of Mansonia humeralis, Ma. cf. titillans, and Ma. amazonensis on the abaxial surface of the leaves of 5 species of aquatic macrophytes, 3 of which have no previous records of association with the oviposition of Mansonia spp., Salvinia molesta Mitch (Salviniales: Salviniaceae), Ludwigia helmintorrhiza (Mart.), and Limnobium spongia (Bosc) Rich. Ex Steud (Alismatales: Hydrocharitaceae), in 3 breeding grounds near the Madeira River in Porto Velho, State of Rondônia.
The aquatic habitats found with Mansonia eggs were characterized for macrophyte species composition, and egg batches were either identified morphologically based on the descriptions of the eggs (Ferreira et al. 2020) or maintained in an insectary until the emergence of adults. Finally, the number of eggs was counted to verify the number of eggs per batch among the different macrophyte species. These values are presented with maximum, minimum, mean, and standard deviation values (Table 1).
Altogether, 13 species of macrophytes were sampled—Eichhornia azurea (Sw.), E. crassipes , Limnobium spongia, Limnobium laevigatum (Humb. and Bonpl. Ex Willd.), Ludwigia helmothorrhiza (Mart.), Ludwigia sedoides (Humb. and Bonpl.) (Alismatales: Hydrocharitaceae), Paspalum repens Bergius (Poales: Poaceae), Cyperus rotundus L. (Poales: Cyperaceae), P. stratiotes, Azolla caroliniana Willd., Salvinia auriculata Aublet complex, Salvinia minima Baker, and Salvinia molesta Mitch. (Salviniales: Salviniaceae), in Luzitânia area (09°03′28.22″S; 64°12′38.52″W), Point Three (09°01′16.46″S; 64°12′21.93″W), and Samauma area (09°11′47.45″S; 64°27′28.02″W). The 3 areas present characteristics of semiperennial lakes, with September being the period with the lowest water level at these points (Fig. 1).
In the Luzitânia area (Fig. 1), 9 species of macrophytes were analyzed: E. crassipes, L. laevigatum, L. spongia, L. helmothorrhiza, L. sedoides, P. repens, C. rotundus, A. caroliniana, and S. molesta. Egg batches were found in S. molesta (n = 125–63.5%), L. helmothorrhiza (n = 13–6.6%), and L. spongia (n = 6–3.0%). The largest number of eggs batches was found in the Luzitânia (n = 144–73.1%). In addition, 2 species of Mansonia were identified, Ma. humeralis and Ma. cf. titillans. The eggs that were found hatched were classified as Mansonia sp. (Table 1).
In Point Three (Fig. 1), 10 species of macrophytes were identified: E. azurea, E. crassipes, L. laevigatum, L. helmothorrhiza, P. repens, C. rotundus, A. caroliniana, S. minima, S. auriculata s.l. and S. molesta. The locality had the second highest number of egg batches (n = 50–25.4%), found in E. crassipes (n = 25–12.7%), L. helmothorrhiza (n = 20–10.2%), and S. molesta (n = 5–2.5%), which were identified as Ma. humeralis, Ma. cf. titillans, and Mansonia sp. (Table 1).
Finally, in Samauma area (Fig. 1), 5 species of macrophytes were identified: E. azurea, E. crassipes, L. laevigatum, P. stratiotes, and S. molesta. In addition, egg batches were found in juvenile P. stratiotes (n = 3–1.5%). The eggs belonged to Ma. amazonensis and Ma. humeralis, and were found in smaller numbers compared to the other studied locations (Table 1).
Salvinia molesta (n = 130–66.0%) presented the highest number of egg batches observed during the study, followed by L. helmothorrhiza (n = 33–16.8%), E. crassipes (n = 25–12.7%), L. spongia (n = 6–3.0%), and P. stratiotes (n = 3–1.5%). The oviposition distance from the leaf edge, measured with a caliper, ranged from 0.1 to 0.3 mm (L. helmothorrhiza), 0.15 to 0.2 mm (P. stratiotes), 0 to 0.39 mm (S. molesta), 0.1 to 0.3 mm (E. crassipes), and 0.1 to 0.2 mm (L. spongia). The egg batches of Ma. amazonensis were deposited 0.15 mm (SD = 0 mm) from the leaf edges of P. stratiotes; in Ma. humeralis, the average distance was 0.18 mm (SD = 0.06 mm) (all macrophytes), and in the case of Ma. cf. titillans, it was 0.10 mm (SD = 0.09 mm) (Table 1).
Results of field collections showed that the highest numbers of egg batches of Ma. humeralis and Ma. cf. titillans were found in S. molesta. In addition, the presence of E. crassipes and P. stratiotes is important for Mansonia larvae and pupae in an aquatic ecosystem (Ronderos and Bachman 1963). Our study suggests that the eggs are deposited in shallow breeding sites, densely covered by small macrophytes, where the leaves are in contact with the water surface in flooded areas but with little influence from the main river flow. These more stable breeding sites ensure that the position of the leaves keeps the eggs trapped in the plant tissue and immersed in water until hatching, for their subsequent migration to macrophytes with more extensive roots, such as E. crassipes and P. stratiotes. In a similar study carried out by Ferreira (1999) on the island of the Marchetaria, the environmental conditions were like that found in this study. However, on Marchetaria Island, egg batches were more frequently collected in P. stratiotes, whereas none was found in Salvinia. Differences observed may indicate either the behavioral plasticity of these species or differences in Mansonia species oviposition preferences.
Laurence and Smith (1958) reported that aquatic grasses such as Azolla, Salvinia, and Lemne are excellent substrates for the oviposition of species of the subgenus Mansonioides in the laboratory. In preliminary studies carried out in our laboratory, engorged field-collected females laid eggs on S. auriculata s.l. and thin styrofoam sheets. Still, they did not lay eggs on E. crassipes and P. stratiotes. This finding can be explained by the position of the young leaves of these macrophytes, which are not in contact with the water surface. However, during field studies, we found Mansonia eggs on old or dead leaves of E. crassipes and P. stratiotes, which were in contact with the water surface; therefore contact with water was an important characteristic of the oviposition sites of the Mansonia species studied.
In addition, the association between Mansonia larvae and the macrophyte roots to obtain oxygen needs to be considered. Field observations indicated that larvae and pupae are found only in the roots of large macrophytes such as E. crassipes and P. stratiotes and more recently in the roots of L. laevigatum (Amorim et al. 2022), possibly due to the availability of oxygen in the plant aerenchyma. Also, the roots of these macrophytes protect them from predators, making it challenging to find the larvae and pupae throughout the roots (Consoli and Lourenço-de-Oliveira 1994).
Breeding site features, such as macrophytes composition, proper positioning of the leaves on the water surface, larger macrophytes to support the fixation of larvae and pupae, and suitable physicochemical parameters of the water, should also be considered as important criteria for Mansonia. Our study not only reinforces the importance of E. crassipes and P. stratiotes as primary host plants for larvae, but also reveals that oviposition can be carried out in other macrophyte species in the presence of the aforementioned environmental criteria.
The authors are grateful for the friendly review of the manuscript by Maria Anice Mureb Sallum. This study was financially supported by the Research and Development project of Santo Antônio Energia (Agência Nacional de Energia Elétrica – ANEEL, “Biomonitoring and Integrated Control of Mansonia Mosquitoes (Diptera: Culicidae) in the region associated with the lake of the Santo Antônio Hydroelectric Power Plant, on the Madeira River, Rondônia, Brazil” project CT.PD.124.2018). In addition, a postdoctoral fellowship was granted to José Ferreira Saraiva by the Fundação para o Desenvolvimento da Universidade Estadual Paulista – FUNDUNESP. We are also grateful for the collection license granted by SISBIO-IBAMA, number 65279-1.
Laboratório de Entomologia Médica do Instituto de Pesquisas Científicas e Tecnológicas do Estado do Amapá – IEPA, Núcleo de Biodiversidade, Rodovia Juscelino Kubitschek, Km-10, CEP 68903-419, Fazendinha, Macapá, Amapá, Brasil.
Programa de Pós-graduação em Biologia Experimental – PGBIOEXP, Campus BR 364, Km 9,5, CEP 76801-059, Porto Velho, Rondônia, Brasil.
Programa de Pós-graduação em Medicina Tropical, Instituto Oswaldo Cruz, FIOCRUZ, Manguinhos, 21040-900, Rio de Janeiro, Brazil.
Santo Antônio Energia – SAE, Rodovia BR365, Km9 (Zona rural), Porto Velho, RO, Brazil.
Laboratório de Fisiologia e Controle de Artrópodes Vetores do Instituto Oswaldo Cruz – FIOCRUZ, Rua Francisco Manuel, No. 102, Bairro Benfica, CEP 20911-270, Rio de Janeiro, Brasil.