RESUMO
Insect cell cultures are an important biotechnological tool for basic and applied studies. The objective of this work was to establish and characterise a new cell line from Culex quinquefasciatus embryonic tissues. Embryonated eggs were taken as a source of tissue to make explants that were seeded in L-15, Grace's, Grace's/L-15, MM/VP12, Schneider's and DMEM culture media with a pH range from 6.7-6.9 and incubated at 28ºC. The morphological, cytogenetic, biochemical and molecular characteristics of the cell cultures were examined by observing the cell shapes, obtaining the karyotypes, using a cellulose-acetate electrophoretic system and performing random amplified polymorphic DNA-polymerase chain reaction analysis, respectively. The Grace's/L-15 medium provided the optimal nutritional conditions for cell adhesion and proliferation. Approximately 40-60 days following the explant procedure, a confluent monolayer was formed. Cellular morphology in the primary cultures and the subcultures was heterogeneous, but in the monolayer the epithelioid morphology type predominated. A karyotype with a diploid number of six chromosomes (2n = 6) was observed. Isoenzymatic and molecular patterns of the mosquito cell cultures matched those obtained from the immature and adult forms of the same species. Eighteen subcultures were generated. These cell cultures potentially constitute a useful tool for use in biomedical applications.
Assuntos
Técnicas de Cultura de Células/métodos , Culex/citologia , Embrião não Mamífero/citologia , Animais , Adesão Celular/fisiologia , Linhagem Celular/química , Linhagem Celular/citologia , Proliferação de Células , Cariótipo , Reação em Cadeia da PolimeraseRESUMO
Insect cell cultures are an important biotechnological tool for basic and applied studies. The objective of this work was to establish and characterise a new cell line from Culex quinquefasciatus embryonic tissues. Embryonated eggs were taken as a source of tissue to make explants that were seeded in L-15, Grace's, Grace's/L-15, MM/VP12, Schneider's and DMEM culture media with a pH range from 6.7-6.9 and incubated at 28ºC. The morphological, cytogenetic, biochemical and molecular characteristics of the cell cultures were examined by observing the cell shapes, obtaining the karyotypes, using a cellulose-acetate electrophoretic system and performing random amplified polymorphic DNA-polymerase chain reaction analysis, respectively. The Grace's/L-15 medium provided the optimal nutritional conditions for cell adhesion and proliferation. Approximately 40-60 days following the explant procedure, a confluent monolayer was formed. Cellular morphology in the primary cultures and the subcultures was heterogeneous, but in the monolayer the epithelioid morphology type predominated. A karyotype with a diploid number of six chromosomes (2n = 6) was observed. Isoenzymatic and molecular patterns of the mosquito cell cultures matched those obtained from the immature and adult forms of the same species. Eighteen subcultures were generated. These cell cultures potentially constitute a useful tool for use in biomedical applications.
Assuntos
Animais , Técnicas de Cultura de Células/métodos , Culex/citologia , Embrião não Mamífero/citologia , Proliferação de Células , Adesão Celular/fisiologia , Linhagem Celular/química , Linhagem Celular/citologia , Cariótipo , Reação em Cadeia da PolimeraseRESUMO
A survey of drainage ditches in suburban areas of La Plata, Buenos Aires province, Argentina for pathogens of Culex pipiens larvae was conducted from 2003 to 2006. C. pipiens larvae of opaque, white color were found in several of those field collections. When the white larvae were dissected and observed by phase-contrast microscopy in wet-mount preparations, the presence of bacteria, located in the hemocoel, was recorded. Laboratory experiments were performed to elucidate the pathway for transmission of this pathogen. Although approaches involving traditional culturing had failed to reveal the identity of the new microorganism present, molecular techniques to identify the pathogen in the studies reported here were successful. The partial sequence of the 16S-rRNA gene constitutes a powerful tool for the detection of new isolates from the hemocoele of C. pipiens larvae. These bacteria were characterized as belonging to the genus Novispirillum. In spite of the genus's wide distribution in different aquatic environments, information related to the parasitic relationship of Novispirillum spp. to aquatic insects is scarce, and this association has not been described in other mosquito species. This report constitutes the first documentation of Novispirillum spp. as a pathogen for mosquito larvae.
Assuntos
Alphaproteobacteria/isolamento & purificação , Culex/microbiologia , Monitoramento Ambiental/métodos , Larva/microbiologia , Alphaproteobacteria/classificação , Alphaproteobacteria/genética , Alphaproteobacteria/ultraestrutura , Animais , Argentina , Culex/citologia , Hemolinfa/microbiologia , Larva/citologia , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Análise de Sequência de RNA , Microbiologia da ÁguaRESUMO
This study describes morphological alterations in the fat body and midgut of Culex quinquefasciatus larvae following exposure to different insecticides. To this end, both third and fourth instars of C. quinquefasciatus larvae were exposed for 30 and 60 min to organophosphate (50 ppb), pyrethroids (20 and 30 ppb), and avermectin derivates (1.5 and 54 ppb). Following incubation, pH measurements of the larvae gut were recorded. The fat body and midgut were also analyzed by light and transmission electron microscopy. These studies demonstrate a decrease in the pH of the larvae anterior midgut following exposure to all of the tested insecticides. Histochemical tests revealed a strong reaction for neutral lipids in the control group and a marked decrease in the group exposed to cypermethrin. Furthermore, a weak reaction with acidic lipids in larvae exposed to deltamethrin, temephos, ivermectin and abamectin was also observed. Insecticide-exposed larvae also exhibited cytoplasm granule differences, relative to control larvae. Finally, we noted a small reduction in microvilli size in the apex of digestive cells, although vesicles were found to be present. The destructive changes in the larvae were very similar regardless of the type of insecticide analyzed. These data suggest that alterations in the fat body and midgut are a common response to cellular intoxication.
Assuntos
Culex/citologia , Culex/efeitos dos fármacos , Corpo Adiposo/citologia , Corpo Adiposo/efeitos dos fármacos , Inseticidas/farmacologia , Animais , Histocitoquímica , Intestinos/citologia , Intestinos/efeitos dos fármacos , Ivermectina/análogos & derivados , Ivermectina/farmacologia , Larva/citologia , Larva/efeitos dos fármacos , Microscopia , Microscopia Eletrônica de Varredura , Piretrinas/farmacologiaRESUMO
Haematophagy, the utilization of blood as food, has evolved independently among insects such as mosquitoes, bedbugs, fleas, and others. Accordingly, several distinct biological adaptations have occurred in order to facilitate the finding, ingestion and digestion of blood from vertebrate sources. Although blood meals are essential for survival and reproduction of these insects, mechanical and chemical stresses are caused by the ingestion of a sizable meal (frequently twice or more times the weight of the insect) containing large amounts of cytotoxic molecules such as haem. Here we present data showing that the stresses caused by a blood meal induce cell death in the midgut epithelium of Culex quinquefasciatus mosquitoes. The process involves apoptosis, ejection of dead cells to the midgut lumen and differentiation of basal regenerative cells to replace the lost digestive cells. The basal cell differentiation in blood-fed mosquito midguts represents an additional mechanism by which insects cope with the stresses caused by blood meals. C. quinquefasciatus adult females are unable to replace lost cells following a third or fourth blood meal, which may have a significant impact on mosquito longevity, reproduction and vectorial capacity.
Assuntos
Apoptose/fisiologia , Culex/citologia , Culex/fisiologia , Trato Gastrointestinal/citologia , Regeneração/fisiologia , Animais , Sangue/metabolismo , Enterócitos/citologia , Enterócitos/fisiologia , Comportamento Alimentar , Feminino , Mucosa Intestinal/ultraestruturaRESUMO
Ultrastructural and cytochemical studies were carried out on spermatid and spermatozoon of the mosquito Culex quinquefasciatus. The spermatozoon appeared threadlike, lacking distinct head and tail regions. The nucleus development involved nuclear shape change and chromatin condensation. The acrosome showed two distinct zones and a subacrosomal space. In the sperm tail an axoneme and two mitochondrial derivatives containing crystalline matrix were observed. The axoneme presented a 9+9+'1' microtubule pattern and the accessory microtubules were composed of fifteen protofilaments. The plasma membrane of the spermatozoon was asymmetric, presenting a 30 nm thick surface coat which was not sensitive to the action of pepsin. Carbohydrates were located in the plasma membrane and in the interface basal layer of the coat, by using the periodic acid-thiosemicarbazide-silver proteinate technique. Lectin gold studies revealed mannose, glucose, galactose, N-acetylglucosamine and sialic acid residues on the cell surface. These carbohydrate residues groups randomly occurred on the surface at the head and tail regions. The surface coat disappeared during the sperm storage in the spermatheca before fertilization.