Diptera: Culicidae, Aedini

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http://dx.doi.org/10.11646/zootaxa.3784.5.8 http://zoobank.org/urn:lsid:zoobank.org:pub:3BC6F0D7-AC54-42CB-BB74-E703F421A41B

Scanning electron microscopy of eggs of Georgecraigius fluviatilis (Lutz) (Diptera: Culicidae, Aedini) JULIANA SOARES SARMENTO1,2, CARLOS BRISOLA MARCONDES3, JERONIMO ALENCAR4, ELIANA MEDEIROS OLIVEIRA5, CECILIA FERREIRA DE MELLO4, VINÍCIOS FERREIRA DE FREITAS3 & JACENIR SANTOS-MALLET1 1

Sector of Medical and Forensic Entomology, Laboratory of Vectors of Leishmaniases, Oswaldo Cruz Institute (Fiocruz), Av. Brasil 4365, CEP 21040-360, Manguinhos, Rio de Janeiro, Brazil. E-mail: [email protected], [email protected] 2 Postgraduate Program on Animal Biology, Institute of Biology, Federal Rural University of Rio de Janeiro, Seropédica, RJ, Brazil 3 Department of Microbiology, Immunology and Parasitology, Centre of Biological Sciences, Federal University of Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil. E-mail: [email protected], [email protected] 4 Diptera Laboratory, Oswaldo Cruz Institute (Fiocruz), Av. Brasil 4365, CEP 21040-360, Manguinhos, Rio de Janeiro, Brazil. E-mail: [email protected], [email protected] 5 Central Laboratory of Electron Microscopy, Federal University of Santa Catarina, 88040-900 Florianópolis, Santa Catarina, Brazil. E-mail: [email protected]

Abstract Scanning Electron Microscopy was used to describe the eggs of Georgecraigius fluviatilis (Lutz). Length is 722.8±39.6 µm and width is 177.1±9.8 µm. Diameter of the micropylar disk, surrounded by an irregular flattened collar, is 28 µm. The outer chorionic sculpture consists of cells of irregular shapes, containing tubercles with pitted surface. In the ventral region, tubercles of several diameters are irregularly distributed in chorionic cells, while in the dorsal region one larger tubercle is surrounded by several smaller ones. The eggs appear to lack structures for adhesion, certainly unnecessary due to the habit of laying eggs separately on water surfaces. Key words: Egg, Ultrastructure, Morphology

Introduction The study of egg morphology is useful for the identification of material from aquatic habitats. Georgecraigius fluviatilis (Lutz) (=Aedes fluviatilis) is widely distributed from southern Mexico to northern (Zavortink 1972, Pecor et al. 2002) and western Argentina (Almirón et al. 1996), and is also recorded in two southernmost Brazilian states, Rio Grande do Sul (Cardoso et al. 2005) and Santa Catarina (Marcondes et al. 2006). Immature forms of Gc. fluviatilis have been found mostly in rock crevices situated near or in rivers (Forattini 1965, Marcondes et al. 2006). They can be found in artificial containers, like tyres and flower pots in cemeteries (Lopes 2002), sometimes with Stegomyia aegypti (Linnaeus) (=Aedes aegypti) (Lopes et al. 1993), which seems to displace them (Lopes 2002). When the eggs of several groups of mosquitoes have finally been described, they can also be used in phylogenetic analyses. Due to the small number of eggs currently described, Motta et al. (2007) did not include egg characteristics in their phylogenetic analysis of Wyeomyia (Sabethini). Additionally, in Reinert et al.'s (2009) phylogenetic analysis of the Aedini, only four morphological characters of the 336 were from eggs, which were unknown for many species. Georgecraigius fluviatilis seems to prefer biting birds (Consoli & Williams 1981), and in a study utilizing several baits (human, chicken, lizard and horse), horses seemed to be more attractive (Neves & Silva 1974), but the number of collected mosquitoes was very small. The species is a very good experimental vector for Plasmodium gallinaceum (Camargo et al. 1983) and has been frequently utilized for experimental studies (e.g., Camargo et al. 1983, Rocha et al. 2004), having been shown to be a

Accepted by G. Courtney: 7 Feb. 2014; published: 1 Apr. 2014

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good experimental vector of yellow fever virus (Davis & Shannon 1931) and suspected vector of this virus in the Valley of Chanaan, state of Espírito Santo, Brazil (Soper et al. 1933). It is a poor experimental vector of Dirofilaria immitis (Leidy) (Kasai & Williams 1986) and of Plasmodium juxtanucleare Versiani & Gomes (Lourenço-de-Oliveira & Castro 1991). The knowledge of egg external surface can also be useful for observation of possible effects of substances potentially useful for control, like garlic extracts (Jarial 2001). Differences between eggs of long-day and short-day Gc. epactius (Dyar & Knab) and Gc. atropalpus (Coquillett) (Linley & Craig Jr. 1994) emphasize another use of the study of morphology of mosquito eggs. Eggs of Gc. fluviatilis, besides some observations on eclosion (Consoli & Williams 1981), general shape (SantosNeto & Marques 1996), and a SEM photograph shown by Forattini (2002, p. 801), have not been studied morphologically. The objective of the present work is to describe the eggs of Gc. fluviatilis in detail, to allow clear differentiation from the eggs of other mosquitoes in the same habitats and possible use of egg characters in phylogenetic studies.

Materials and methods Eggs of Gc. fluviatilis used in this study were obtained from blood-fed females captured in the Ecological Guapiçu Reserve, located in the Cachoeiras de Macacu City in the Brazilian state of Rio de Janeiro (22o25'23''S 42o44'30''W). The females were captured with an oral suction tube (Marcondes et al. 2007) and transported in same day to the laboratory. Mosquitoes were maintained in 30 ml plastic vials for 4-5 days until they laid eggs. Eggs were fixed in 2.5% glutaraldehyde and post-fixed in 1% osmium tetroxide, both buffered with 0.1M sodium cacodylate at pH 7.2. After washing in the same buffer, the eggs were dehydrated in a series of increasing concentrations of ethanol and subjected to the critical-point drying method using superdry CO2 in Balzers apparatus. The eggs were then mounted on metal supports, gold plated and observed and photographed in a JEOL JSM 6390LV scanning electron microscope (Akishima, Tokyo, Japan), at magnifications of 200 to 5,000 times. Measurements were made directly on the images obtained, with the aid of the Semafore digital slow scan image recording system, version 3.1© (Insinooritoi misto J. Rimppi Oy, Finland), and analyzed using a SEM Control User Interface version 8.24© (JEOL TECHNICHS LTD), coupled to the microscope. Measurements taken were total length, total width and diameter of chorionic tubercles, the micropyle and its annexes. The terminology for describing the eggs is that of Harbach and Knight (1980). Mosquito genera and subgenera are abbreviated according to Reinert (2009). Ochlerotatus, Georgecraigius, Gymnometopa and Stegomyia were accepted as genera (Reinert 2000, Marcondes 2007, Reinert et al. 2008).

Results Eggs of Gc. fluviatilis (length: 722.8±39.6 µm; width 177.1±9.8 µm; n=8, length/width: 4.8±3.7 µm) are cigar shaped, widest at anterior third and have the ventral surface more convex than the dorsal suface (Fig. 1A). The micropyle is surrounded by a micropylar disk and by a complete and irregular flattened micropylar collar (Fig. 1B). The outer chorionic sculpture consists of cells of irregular shapes, each containing 27.2±5.9 µm tubercles of 3.2±0.57 µm (n =12) in the cells of the ventral surface (Fig. 1C). Chorionic cells tend to contain several tubercles of different diameters in the ventral region, while those in dorsal region include one large and several smaller tubercles around the central one, constituting an irregular lozenge or elongate hexagon (Fig. 1D) in the ventral region. The diameter of larger tubercles is 2.65±0.41 µm and the smaller ones 1.4±0.24 µm. In the dorsal region, the diameter of central tubercles is 6.04±0.61 µm and the smaller ones 1.6±0.29 µm. In the transition between dorsal and ventral regions, the diameter of central tubercles is 5.1±0.58 µm and the smaller ones 2.0±0.64 µm (Fig. 1E). The upper surface of the larger tubercles is pitted (Fig. 1D). Structures related to adhesion are absent.

Discussion Eggs of Gc. fluviatilis are usually deposited singly and directly on the water (Cônsoli & Williams, 1976), different to the eggs of St. aegypti and St. albopicta (Skuse) (=Aedes albopictus), which are frequently adhered to surfaces (Consoli & Oliveira 1993). Linley (1989), in his detailed description of eggs of the last two species, did not cite structures

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associated to adhesion to surfaces. The absence of filaments like those reported for Haemagogus equinus Theobald and Hg. janthinomys Dyar by Linley & Chadee (1991) was not considered relevant to eggs of Ochlerotatus terrens (Walker), because they could have been replaced by elongate fusiform tubercles (Alencar et al. 2005). The absence of any similar structure in Gc. fluviatilis agrees with the observed oviposition habit of this mosquito. Santos-Neto & Marques (1996), measuring a small number of eggs of Gc. fluviatilis collected in the north-east of São Paulo state, obtained 675 µm and 180 µm for means of total length and maximum width respectively, slightly shorter than eggs measured in the present study. Forattini (2002, p. 801) showed a SEM photograph of part of an egg of Gc. fluviatilis, probably from the dorsal region, based on present observations (Fig. 1D).

FIGURE 1. Eggs of Georgecraigius fluviatilis. A—anterior end at top of egg; B—micropyle surrounded by irregular flattened micropylar collar; C—cells of irregular shapes on ventral surface; D—details of chorionic cells of dorsal region showing one large and several smaller tubercles around the central tubercle; E—transition between dorsal and ventral regions.

Immature forms of Gc. fluviatilis were found with those of Anopheles argyritarsis Robineau-Desvoidy, Culex coronator Dyar & Knab, Cx. nigripalpus Theobald, Cx. erraticus (Dyar & Knab), and Cx. mutator Dyar & Knab in Belize (Pecor 2002). Since all these species have lateral floats (Harbach & Knight 1980) or are in rafts, they are easily distinguished from Gc. fluviatilis eggs. It would be very useful to always report the simultaneous finding of different species in each water collection. Immature forms of Gc. fluviatilis were the only ones found in rock crevices in the middle of a river in the west of Santa Catarina, but were not found in those near the edges, partially in shadow and with leaves, where only predatory species (Toxorhynchites sp. and Lutzia bigoti (Bellardi)) were found (Marcondes et al. 2006). Immature forms of Gc. fluviatilis were recently obtained in a plastic vial with water in the fire scape area of the third floor (27o35’54.14”S 48o30’55.33”W), near the CBM laboratory, in the same place where, some years ago, those of Oc. scapularis (Rondani) were obtained from a small asbestos cement water tank. In the Brazilian state of Paraná, Gc. fluviatilis was the third most abundant species in a 500 litre asbestos cement water tank, with at least nine other species (Lopes 2002). Eggs of all these species, except Cx. coronator, Cx. eduardoi Casal & Garcia and Cx. saltanensis Dyar, had previously been described, at least by light microscopy, but eggs of these species of Culex occur in rafts and are thus easily distinguished from those of Gc. fluviatilis. The pitted upper surface of tubercles of Gc. fluviatilis eggs is similar to that observed in Gimnometopa mediovittatus (Coquillett) (=Aedes mediovittatus) eggs (Linley & Clark 1989). The function of such pits should be investigated. Besides Gc. fluviatilis of subgenus Horsfallius, the genus includes two other species in subgenus SEM OF EGGS OF GEORGECRAIGIUS FLUVIATILIS

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Georgecraigius, Gc. epactius and Gc. atropalpus, that are distributed in Central and North America. Both have had their exochorial surface briefly described and illustrated (Burst, 1974), showing a quite different pattern of distribution of tubercles and cells compared to Gc. fluviatilis. Egg surface was added to crossings among species on this genus for the definition of species and subspecies, separating Gc. epactius from Gc. atropalpus and synonymizing Ae. atropalpus nielseni O’Meara & Craig Jr. and Ae. atropalpus pericharis Dyar with Gc. epactius (Burst 1974). Munstermann (1980) observed enzyme variation among populations of Gc. epactius and Gc. atropalpus, and studies on their eggs and the eggs of Gc. fluviatilis could be very useful. Zavortink (1972) cited differences in male genitalia of Gc. fluviatilis, comparing mosquitoes from the Guianas, extreme south of Brazil (in reality, he examined material from states in the south-east, and not from any of three states of southern Brazil) and adjacent northern Argentina to those from other regions. Franca, state of São Paulo, south-east of Brazil, is the type locality of Gc. fluviatilis. Horsfal et al. (1970) cited variations between eggs of Oc. sticticus (Meigen) (=Aedes sticticus) and Aedimorphus vexans (Meigen) (=Aedes vexans) collected in different localities in North America. It would be advisable to check the variation in widely distributed species, like Gc. fluviatilis, utilizing egg characters as a tool for the characterization of possible species complexes. Georgecraigius fluviatilis is easily reared in the laboratory (Consoli & Williams 1976, 1981), having been frequently used for studies of bird malaria, and it would be relatively easy to get eggs from several regions for comparative studies.

Acknowledgements We acknowledge the financial support by Fundação Carlos Chagas de Amparo à Pesquisa no Estado do Rio de Janeiro FAPERJ (E26-103.035/2012 and 112.076/2012) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (475256/2011-5). To Rudolf Barth Electron Microscopy Platform for allowing the use of the scanning electron microscope. Dr. Ralph Harbach (NHM, London) emphasized some pertinent references. Dr. Gustavo Rossi (CEPAVE, Buenos Aires) sent useful information on Culex eggs. The authors also thanks Nicolas and Raquel Locke as president and vice president, and Jorge Bizarro as research coordinator of REGUA, for the facilities granted for carrying out the studied.

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