Vitellogenin genes are transcribed in Culex quinquefasciatus ovary.

BACKGROUND: Culex quinquefasciatus, a cosmopolitan, domestic, and highly anthropophilic mosquito, is a vector of pathogenic arboviruses such as West Nile virus and Rift Valley virus, as well as lymphatic filariasis. The current knowledge on its reproductive physiology regarding vitellogenin expression in different tissues is still limited. OBJECTIVES: In this study, we analysed the transcriptional profiles of vitellogenin genes in the fat body and ovaries of C. quinquefasciatus females during the first gonotrophic cycle. METHODS: C. quinquefasciatus ovaries and/or fat bodies were dissected in different times during the first gonotrophic cycle and total RNA was extracted and used for reverse transcription polymerase chain reaction, quantitative real time-PCR, and in situ hybridisation. FINDINGS: We confirmed the classical descriptions of the vitellogenic process in mosquitoes by verifying that vitellogenin genes are transcribed in the fat bodies of C. quinquefasciatus females. Using RNA in situ hybridisation approach, we showed that vitellogenin genes are also transcribed in developing ovaries, specifically by the follicle cells. MAIN CONCLUSIONS: This is the first time that vitellogenin transcripts are observed in mosquito ovaries. Studies to determine if Vg transcripts are translated into proteins and their contribution to the reproductive success of the mosquito need to be further investigated.

Vitellogenin genes are transcribed in Culex quinquefasciatus ovary

BACKGROUND Culex quinquefasciatus, a cosmopolitan, domestic, and highly anthropophilic mosquito, is a vector of pathogenic arboviruses such as West Nile virus and Rift Valley virus, as well as lymphatic filariasis. The current knowledge on its reproductive physiology regarding vitellogenin expression in different tissues is still limited. OBJECTIVES In this study, we analysed the transcriptional profiles of vitellogenin genes in the fat body and ovaries of C. quinquefasciatus females during the first gonotrophic cycle. METHODS C. quinquefasciatus ovaries and/or fat bodies were dissected in different times during the first gonotrophic cycle and total RNA was extracted and used for reverse transcription polymerase chain reaction, quantitative real time-PCR, and in situ hybridisation. FINDINGS We confirmed the classical descriptions of the vitellogenic process in mosquitoes by verifying that vitellogenin genes are transcribed in the fat bodies of C. quinquefasciatus females. Using RNA in situ hybridisation approach, we showed that vitellogenin genes are also transcribed in developing ovaries, specifically by the follicle cells. MAIN CONCLUSIONS This is the first time that vitellogenin transcripts are observed in mosquito ovaries. Studies to determine if Vg transcripts are translated into proteins and their contribution to the reproductive success of the mosquito need to be further investigated.

Two cathepsins B are responsible for the yolk protein hydrolysis in Culex quinquefasciatus.

Despite the established role of Culex quinquefasciatus as a vector of various neurotropic viruses, such as the Rift Valley and West Nile viruses, as well as lymphatic filariasis, little is known regarding the organism's reproductive physiology. As in other oviparous animals, vitellogenin, the most important source of nutrients for the embryo development, is digested by intracellular proteases. Using mass spectrometry, we have identified two cathepsin B homologues partially purified by self-proteolysis of Cx. quinquefasciatus total egg extract. The transcriptional profile of these two cathepsin B homologues was determined by quantitative RT-PCR, and the enzymatic activity associated with the peptidase was determined in ovaries after female engorgement. According to the VectorBase (vectorbase.org) annotation, both cathepsin B homologues shared approximately 66% identity in their amino acid sequences. The two cathepsin B genes are expressed simultaneously in the fat body of the vitellogenic females, and enzymatic activity was detected within the ovaries, suggesting an extra-ovarian origin. Similar to the transcriptional profile of vitellogenin, cathepsin B transcripts were shown to accumulate post-blood meal and reached their highest expression at 36 h PBM. However, while vitellogenin expression decreased drastically at 48 h PBM, the expression of the cathepsins increased until 84 h PBM, at which time the females of our colony were ready for oviposition. The similarity between their transcriptional profiles strongly suggests a role for the cathepsin B homologues in vitellin degradation.

Culex quinquefasciatus storage proteins.

Insect storage proteins accumulate at high levels during larval development of holometabolous insects. During metamorphosis they are degraded, supplying energy and amino acids for the completion of adult development. The genome of Culex quinquefasciatus contains eleven storage protein-coding genes. Their transcripts are more abundant in larvae than in pupae and in adults. In fact, only four of these genes are transcribed in adults, two of which in blood-fed adult females but not in adult males. Transcripts corresponding to all Cx. quinquefasciatus storage proteins were detected by RT-PCR, while mass spectrometric analysis of larval and pupal proteins identified all storage proteins with the exception of one encoded by Cq LSP1.8. Our results indicate that the identified Cx. quinquefasciatus storage protein-coding genes are candidates for identifying regulatory sequences for the development of molecular tools for vector control.

Effects of Wolbachia on fitness of Culex quinquefasciatus (Diptera; Culicidae).

Wolbachia are α-proteobacteria that were first reported in Culex pipiens mosquitoes early in the twentieth century. Since then, the effect of Wolbachia on their host's reproduction has drawn attention and has been increasingly investigated. Given the extreme complexity of this interaction, new study cases are welcomed to enhance its understanding. The present work addressed the influence of Wolbachia on Cx. quinquefasciatus, the cosmopolitan member of the Cx. pipiens complex. Samples of a Cx. quinquefasciatus colony (wPip(+)) originated from individuals naturally infected by Wolbachiapipientis B strain, were cured with tetracycline, yielding a Wolbachia-free colony (wPip(-)). Both the presence of bacteria and the efficiency of bacterial elimination were checked by PCR of the wsp gene. Total reproductive unidirectional incompatibility occurred when wPip(-) females were crossed with wPip(+) males, whereas the other three types of reciprocal crosses were viable. Reproductive aspects were also comparatively evaluated between colonies. Concerning oviposition time during the first gonotrophic cycle, wPip(+) females developed and laid eggs earlier than did wPip(-) females. Reproductive fitness was higher among wPip(-) than wPip(+) females regarding the following parameters: fertility: egg rafts/fed females; fecundity: eggs/raft, and viability: larvae/eggs. Conversely, longevity of wPip(-) females was lower. Summarising, although the infected mosquitoes have the advantage of a higher longevity, they have lower reproductive fitness. Our results are partly distinct from all other reports on Aedes and Culex mosquitoes previously published.

Effects of Wolbachia on fitness of Culex quinquefasciatus (Diptera Culicidae)

Wolbachia are á-proteobacteria that were first reported in Culex pipiens mosquitoes early in the twentieth century. Since then, the effect of Wolbachia on their host’s reproduction has drawn attention and has been increasingly investigated. Given the extreme complexity of this interaction, new study cases are welcomed to enhance its understanding. The present work addressed the influence of Wolbachia on Cx. quinquefasciatus, the cosmopolitan member of the Cx. pipiens complex.

Culex quinquefasciatus vitellogenesis: morphological and biochemical aspects.

The vitellogenic process in Culex quinquefasciatus, which is triggered by a blood meal, involves the synthesis, distribution and storage of the nutrients necessary for embryo development. The fat body of an adult female Cx. quinquefasciatus revealed two cell types: large trophocytes and small, eosinophilic, "oenocyte-like" cells, which show no morphological changes throughout the gonotrophic cycle. Trophocytes, which only begin to synthesise vitellogenin (Vg) 12 h post-blood meal (PBM), undergo a series of morphological changes following engorgement. These changes include the expansion of the rough endoplasmic reticulum (RER) and Golgi complex, which are later destroyed by autophagosomes. At 84 h PBM, trophocytes return to their pre-engorgement morphology. The ovarian follicles of non-blood-fed Cx. quinquefasciatus contain a cluster of eight undifferentiated cells surrounded by follicular epithelium. After engorgement, the oocyte membrane facing the perioocytic space increases its absorptive surface by microvilli development; large amounts of Vg and lipids are stored between 24 and 48 h PBM. Along with yolk storage in the oocyte, follicular cells exhibit the development of RER cisternae and electron-dense granules begin to fill the perioocytic space, possibly giving rise to endochorion. Later in the gonotrophic cycle, electron-dense vesicles, which are possible exochorion precursors, fuse at the apical membrane of follicular cells. This fusion is followed by follicular cell degeneration.

Culex quinquefasciatus vitellogenesis: morphological and biochemical aspects

The vitellogenic process in Culex quinquefasciatus, which is triggered by a blood meal, involves the synthesis, distribution and storage of the nutrients necessary for embryo development. The fat body of an adult female Cx. quinquefasciatus revealed two cell types: large trophocytes and small, eosinophilic, "oenocyte-like" cells, which show no morphological changes throughout the gonotrophic cycle. Trophocytes, which only begin to synthesise vitellogenin (Vg) 12 h post-blood meal (PBM), undergo a series of morphological changes following engorgement. These changes include the expansion of the rough endoplasmic reticulum (RER) and Golgi complex, which are later destroyed by autophagosomes. At 84 h PBM, trophocytes return to their pre-engorgement morphology. The ovarian follicles of non-blood-fed Cx. quinquefasciatus contain a cluster of eight undifferentiated cells surrounded by follicular epithelium. After engorgement, the oocyte membrane facing the perioocytic space increases its absorptive surface by microvilli development; large amounts of Vg and lipids are stored between 24 and 48 h PBM. Along with yolk storage in the oocyte, follicular cells exhibit the development of RER cisternae and electron-dense granules begin to fill the perioocytic space, possibly giving rise to endochorion. Later in the gonotrophic cycle, electron-dense vesicles, which are possible exochorion precursors, fuse at the apical membrane of follicular cells. This fusion is followed by follicular cell degeneration.