Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 97, Num. 4, 2002, pp. 589-596

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 97(4) 2002, pp. 589-596

Anopheles albitarsis Embryogenesis: Morphological Identification of Major Events

Adelaide Tardin Monnerat, Marcelo Pelajo Machado*, Bruno Silva Vale*, Maurilio José Soares**, José Bento Pereira Lima***/****, Henrique Leonel Lenzi*, Denise Valle***/****/^+
+Corresponding author. Fax: +55-21-2573.4468. E-mail: dvalle@ioc.fiocruz.br

Departamento de Bioquímica e Biologia Molecular *Departamento de Patologia **Departamento de Ultra-estrutura e Biologia Celular ***Departamento de Entomologia, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil ****Laboratório de Entomologia, Instituto de Biologia do Exército, Rio de Janeiro, RJ, Brasil

This investigation received financial assistance from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, Conselho Nacional de Desen-volvimento Científico e Tecnológico, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro and Fundação Oswaldo Cruz.

Received 22 October 2001
Accepted 28 January 2002

Code Number: oc02112

Anopheles albitarsis embryogenesis was analyzed through confocal microscopy of clarified eggs. Using Drosophila melanogaster as reference system, the major morphogenetic events (blastoderm, gastrulation, germ band extension, germ band retraction, dorsal closure) were identified. The kinetics of early events is proportionally similar in both systems, but late movements (from germ band retraction on) progress slower in An. albitarsis. Major differences in An. albitarsis related to D. melanogaster were: (1) pole cells do not protrude from the blastoderm; (2) the mosquito embryo undergoes a 180º rotation movement, along its longitudinal axis; (3) the head remains individualized throughout embryogenesis; (4) extraembryonary membranes surround the whole embryo. A novel kind of malaria control is under development and is based on the use of genetically modified mosquitoes. Phenotypic analysis of the embryonic development of mutants will be imposed as part of the evaluation of effectiveness and risk of employment of this strategy in the field. In order to accomplish this, knowledge of the wild type embryo is a prerequisite. Morphological studies will also serve as basis for subsequent development biology approaches.

Key words: Anopheles - malaria vector - embryo - development - mosquito

Nowadays, nearly 30% of the world population, mainly in tropical countries, is at risk of acquiring malaria, an endemic disease that kills 1-2 million people each year (Buttler et al. 1997, Morel 2000). Malaria is caused by protozoan parasites belonging to the genus Plasmodium which undergo an obligatory part of their life cycle inside a mosquito. A major obstacle to control of human malaria is the varied degree of resistance exhibited by Plasmodium strains and Anopheles wild populations to the presently available chemotherapics and insecticides, respectively (Greenwood 1997).

An alternative strategy to malaria vector control is presently under way and involves the construction of genetically modified mosquitoes, refractory to Plasmodium infections (Kidwell & Ribeiro 1992, Crampton 1994, Buttler et al. 1997). Although most of the present studies aiming the construction of transgenic mosquitoes rely on molecular assays (Coates et al. 1998, Jasinskiene et al. 1998, Catteruccia et al. 2000), knowledge of the basic developmental biology of vectors (and of their embryogenesis in particular) is also of fundamental importance when the establishment of transformants is concerned (Bate & Arias 1993). In order to generate transformed lineages, exogenous DNA must be injected into embryos at a very specific position and time during their development (Miller et al. 1987).

It is expected that several mutants and transgenic lines will be obtained by the introduction and mobilization of transposable elements into Anopheles spp. Phenotypic analysis of these mutants is imposed as part of the evaluation of effectiveness and risk the use, in the field, of a vector control strategy based on transgenic mosquitoes. Since, as it occurs with the model system D. melanogaster, several mutants affecting viability are expected to be embryonic lethal (Bate & Arias 1993), knowledge of the morphological alterations in embryos belonging to mutant mosquito lineages will be an important component in their phenotypic analysis. Furthermore, in cases where mutants are not available, the study of a gene's expression pattern in a wild type embryo can contribute to elucidate its function and, consequently, define its relevance for the viability (and control) of a defined organism.

It turns out that a systematic analysis of the embryonic development of wild type mosquitoes is important in order to accomplish exogenous DNA injections, phenotypic analysis of mutants and even to define the expression pattern of new genes. However, this has been a neglected issue: so far only embryos of a single Anopheles species have been submitted to a morphological analysis (An. maculipennis, Ivanova-Kazas 1949). Information on other mosquito embryos, mainly obtained in the 60's and 70's, is often scarce and incomplete. Among mosquitoes, mainly Culex and Aedes embryos have been investigated (Rosay 1959, Davis 1967, Guichard 1971, Raminani & Cupp 1975, 1978).

In the present study we have identified, by comparison with D. melanogaster, the major morphological events occurring throughout the embryonic development of a neotropical malaria vector, An. (Nyssorhynchus) albitarsis. This corresponds to the first step towards a systematic morphological analysis of wild type Anopheles embryos aiming to contribute to the establishment of new vector control methods and to the further analysis of expression of different genes during development in these species.

MATERIALS AND METHODS

Insects - An. albitarsis sensu stricto females came from a free mating stable colony maintained in the laboratory since 1995 (Horosko et al. 1997), kept at 26ºC and 80% r. h. Larvae were fed with powdered fish food (1:1 mixture of Tetramin Baby Fish Type L and Type E) and adults were constantly supplied with 10% sucrose solution. Females starved for 2-4 h were fed on guinea pigs in order to elicit eggs production. D. melanogaster embryos were obtained from wild type stocks kindly provided by Drs Ricardo Ramos and Antônio Bernardo Carvalho (Universidade Federal do Rio de Janeiro).

Mosquito eggs collection - Collection of the eggs was done at 2 h intervals, from egglaying to hatching. In order to overcome the crepuscular oviposition, typical of An. albitarsis, some time points were collected according to the method described by Lanzara et al. (1988). This method consists in the induction of oviposition through removal of one wing from females that have been blood fed four days before. The mosquitoes were then placed individually in small plastic cups filled with dechlorinated water and maintained in the dark. After oviposition the females were removed and the eggs were kept in the plastic cups for defined periods of time. Collection of eggs for shorter periods of time was not possible due to the extreme sensitivity of An. albitarsis to manipulation.

Mosquito eggs clarification - Eggs chorion clarification (also corresponding to embryo development interruption) has been performed in small glass vials filled with 0.037 M sodium chlorite plus 1.45 M acetic acid, tightly closed and kept under gentle agitation, protected from light, at room temperature (Tripš 1970). The clarification procedure was monitored under a stereomicroscope, and varied from 42 to 66 h. The eggs were then washed five times with phosphate buffered saline pH 7.5 and wholemounted in slides in a 1:1 glycerol:phosphate buffered saline solution.

Mosquito embryo analysis - Embryos were visualized by confocal microscopy, performed with a Zeiss LSM 410 using a He/Ne laser beam (543 nm). Autofluorescence signal was achieved with a LP 570 filter (Valle et al. 1999).

Drosophila embryos - Staged D. melanogaster embryos were submitted to the Forbes and Ingham (1993) protocol used to remove all eggshell layers. The embryos were then fixed for 1 h with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, washed in cacodylate buffer, post-fixed for 30 min with 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2, rinsed in buffer, dehydrated in acetone and critical point dried. The eggs were then adhered to scanning electron microscopy stubs covered with adhesive tape, coated with a 20 nm thick gold layer in a sputtering device and observed in a Zeiss DSM 940 scanning electron microscope (SEM).

RESULTS

Mosquito embryos are covered by an eggshell made by a compound exochorion (or "outer" chorion layer) and a homogeneous endochorion ("inner" chorion layer) (Hinton 1968, Valle et al. 1999, Monnerat et al. 1999). Eggshells are bright and soft at oviposition, but eggs of Anopheles, Culex and Aedes spp. soon turn dark and rigid (Clements 1992), a process that precludes the direct observation of the embryos. Darkening and hardening of eggshells are both consequence of sclerotization, a process occurring in the endochorion (Li 1994). Use of the clarification protocol developed by Tripš (1970) allowed to remove the exochorion and to render the endochorion of An. albitarsis transparent, without affecting embryo morphological integrity. This enabled observation of the embryos development, carried out by confocal microscopy. Both the remaining endochorion and the embryos inside were fluorescent, enabling the morphological identification of the major morphogenetic movements occurring during embryogenesis through analysis of the reflected light.

In order to follow the convention adopted for the model system D. melanogaster, pictures are shown here with the anterior pole to the left and, unless otherwise indicated, the dorsal side facing upwards, as previously suggested (Bate & Arias 1993, Valle et al. 1999). Structures and features appearing in the mosquito embryo are localized in terms of percent of egg length (EL), where zero is the posterior pole and the anterior pole corresponds to 100% (Bate & Arias 1993). Staged D. melanogaster embryos were examined by SEM after removal of eggshell layers, and embryos at typical major developmental stages were included in some plates for comparison.

Blastoderm formation - Early D. melanogaster embryos display homogeneously distributed yolk granules; only the periphery of the embryo and a cytoplasmic island (energid) in the anterior third of the egg, surrounding the female pronucleus, are yolk-free. As cleavage divisions take place, each zygotic nucleus becomes surrounded by a cytoplasmic island. In D. melanogaster, the syncytial blastoderm takes place from 1 h 20' to 2 h 10' at 25ºC, when cellularization of the germ lineage, located at the posterior pole, occurs (Fig. 2A). The somatic cells are formed later (2 h 10'-2 h 50') by synchronous extension of membrane furrows along the embryo cortex (Bate & Arias 1993, Campos-Ortega & Hartenstein 1997), characterizing the cellular blastoderm (Fig. 3A).

Early An. albitarsis embryos occupy the whole egg, their cortical cytoplasm being adjacent to the eggshell. The anterior half of the embryo is larger than the posterior side. At 0-2 h after egglaying, several spherical autofluorescent dots of undefined nature are observed scattered throughout the embryo (Fig. 1). Rounded patches exhibiting less intense fluorescence are also visible. At 2-4 h after egglaying, nuclei have already reached the periphery and the yolk mass is concentrated in the embryo internal domain (Fig. 2B), a picture typical of dipteran eggs. Analysis of 2-4 h old embryos at higher magnification (Fig. 2C) confirmed the cortical position of nuclei but did not reveal any sign of cellularization, indicating that, at that time, embryos are in the syncytial blastoderm stage.

At 4-6 h after egglaying, the cellular limits are clearly visible at the An. albitarsis embryo cortex (Fig. 3B, C). Cells of two distinct sizes are observed: while cells with surface area of about 80 µm² are concentrated in the posterior pole, those measuring 20-40 µm² can be found in the rest of the embryo. These smaller cells correspond to the somatic lineage and will give rise to all body compartments. The larger cells are supposed to be the pole cells, precursors of the germ line. These morphological characteristics are typical of the cellular blastoderm stage.

Gastrulation - D. melanogaster early gastrulation is a short stage (10'), characterized by the invagination of mesoderm and endoderm and by the appearance of a cephalic furrow, at around 65% EL (Fig. 4A, Campos-Ortega & Hartenstein 1997).

In An. albitarsis, at 4-6 h after oviposition some embryos develop a lateral invagination at 70% EL (compare panels A and B of Fig. 4, corresponding to D. mela-nogaster and An. albitarsis, respectively). This was interpreted as the first sign of gastrulation, typified by the formation of the cephalic furrow. In some cases a longitudinal ventral enlargement was detected in the trunk portion of the embryo that should correspond to the beginning of mesoderm invagination (visible in Drosophila, at the bottom of Fig. 4A).

Germ band extension - Germ band extension in Diptera is characterized by folding the trunk domain to the dorsal side of the embryo and its further progression towards the anterior portion, as illustrated in Fig. 5A. In D. melanogaster embryos, this stage is divided into a rapid early phase, when the germ band extends until 60% EL, in 30 min, and a slow phase, that requires additional 2 h to extend 15% further (up to 75% EL, Campos-Ortega & Hartenstein 1997).

An. albitarsis germ band extension is first detectable around 8 h after egglaying. Fig. 5B shows a 6-8 h old embryo, in lateral position, where the germ band extends towards the anterior pole. A rate difference between the early and the late phases of germ band extension was also observed in this species: while 4-6 h old embryos are at cellular blastoderm or gastrulation (Figs 3, 4), 6-8 h old ones have already proceeded germ band extension until 60% EL (Fig. 5B). By contrast, the maximal germ band extension (70% EL) takes another 2 h (8-10 h old embryos, Fig. 5C) to be accomplished.

The first external signs of segmentation appear at 10-12 h after egglaying, when the embryo is still at the maximal germ band extension stage (Fig. 5D). In opposition to Drosophila embryos, where segmentation is detected simultaneously in the whole body, in An. albitarsis the first external metameric units are initially observed only at the anterior region.

During germ band extension, the embryo starts to detach itself from the eggshell, a feature clearly visible at the end of this stage (10-12 h old embryos, Fig. 5D). In some cases the original egg shape is slightly altered, as a consequence of its softening by the clarification procedure.

Germ band retraction - After the complete extension of the germ band and appearance of the initial segmentation signs, the embryo starts the germ band retraction, a process that establishes the normal anatomical aspect of the larva (Fig. 6A). In An. albitarsis, germ band retraction is first detected at 14-16 h after egglaying (Fig. 6B). During this stage, the intersegmental furrows deepen, rendering the segments more and more visible throughout the embryo body (compare Figs 6B-D). The germ band posterior tip can be detected at 45 and 30% EL at, respectively, 14-16 h (Figs 6B, 6C) and 16-18 h (Fig. 6D) of development. During this phase the stomodeum, that will give rise to the anterior gut, is clearly visible at the ventral side of the anterior pole (arrowhead on Fig. 6C). The ventral nerve cord invagination occurring in the embryo trunk is also identified, appearing as a more intensely fluorescent longitudinal stripe, when the embryo is at a dorso-lateral (Fig. 6C) or dorsal (Fig. 6D) position.

Es, knowledgelopmnrulatr> Accepted 28 January 2002

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RESULTS

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