Males of Aedes aegypti show different clock gene expression profiles in the presence of conspecific females.

BACKGROUND: The study of behavioral and physiological traits in mosquitoes has been mainly focused on females since males are not hematophagous and thus do not transfer the parasites that cause diseases in human populations. However, the performance of male mosquitoes is key for the expansion of populations and the perpetuation of mosquito species. Pre-copulatory communication between males and females is the initial and essential step for the success of copulation and studying the male facet of this interaction provides fertile ground for the improvement of vector control strategies. Like in most animals, reproduction, feeding, and oviposition are closely associated with locomotor activity in mosquitoes. Rhythmic cycles of locomotor activity have been previously described in Aedes aegypti, and in females, they are known to be altered by blood-feeding and arbovirus infection. In previous work, we found that males in the presence of females significantly change their locomotor activity profiles, with a shift in the phase of the activity peak. Here, we investigated whether this shift is associated with changes in the expression level of three central circadian clock genes. METHODS: Real-time PCR reactions were performed for the gene period, cycle, and cryptochrome 2 in samples of heads, antennae, and abdominal tips of solitary males and males in the presence of females. Assays with antennae-ablated males were also performed, asking whether this is an essential organ mediating the communication and the variation in activity profiles. RESULTS: The gene period showed a conserved expression pattern in all tissues and conditions, while the other two genes varied according to the male condition. A remarking pattern was observed in cry2, where the difference between the amplitude of expression at the beginning of photophase and the expression peak in the scotophase was greater when males were in the presence of females. Antennae ablation in males did not have a significant effect on the expression profiles, suggesting that female recognition may involve other senses besides hearing and olfaction. CONCLUSION: Our results suggest that the expression of gene cryptochrome 2 varies in association with the interaction between males and females.

Bases genéticas e regulatórias da variação no perfil de atividade de voo associada à reprodução em machos de Aedes aegypti / Genetic and regulatory bases of the variation in the flight activity profile associated with reproduction in males of Aedes aegypti

O estudo de características comportamentais e fisiológicas em mosquitos tem sido feito principalmente em fêmeas, já que os machos não são hematófagos e por isso não são vetores de parasitas que causam doenças na população humana. No entanto, o estudo do comportamento reprodutivo de machos é de grande importância, já que dele depende o sucesso reprodutivo e a perpetuação da espécie. O reconhecimento e a corte configuram a comunicação pré-cópula entre machos e fêmeas e são etapas essenciais para que a cópula seja bem-sucedida. Portanto, qualquer fator que altere o reconhecimento pré-cópula pode afetar o crescimento populacional dessas espécies. O conhecimento acerca desse tema tem potencial de gerar informações úteis para o avanço de estratégias de controle de vetores. Funções como reprodução, alimentação e oviposição estão associadas com a atividade locomotora, e algumas dessas funções ocorrem em ciclos rítmicos. Quando estes ciclos se mantêm em condições de claro/escuro e escuro constante, estão sob a regulação de um marca-passo endógeno, chamado de relógio circadiano. Em Araripe et al. (2018), nosso grupo estudou o comportamento de machos de Aedes aegypti e foi encontrado que os machos têm sua atividade locomotora significativamente alterada quando expostos a fêmeas virgens confinadas.

A partir disso, os objetivos deste projeto foram investigar se a alteração comportamental observada em machos expostos a fêmeas está associada com alterações em nível de expressão de alguns dos principais genes do relógio circadiano e genes envolvidos com a percepção olfativa e com o comportamento reprodutivo, ao longo de 24 horas (LD12:12). Reações de PCR Quantitativo em Tempo Real foram realizadas para os genes period, cycle, cryptocrhome 2, Orco e fruitless em amostras de cabeças, antenas e pontas de abdômen de machos nas seguintes condições: "machos sozinhos", "machos com fêmeas", "machos sem antenas sozinhos" e "machos sem antenas com fêmeas". O gene period apresentou um padrão de expressão conservado em todas os tecidos e condições. O gene cycle apresentou uma variação no pico de expressão, dentro da fase clara, em todos tecidos, enquanto o vale foi bem definido. O gene cryptochrome 2 apresentou diferenças entre as condições, sugerindo que a presença de fêmeas tem efeito sobre a expressão. Em cabeças, pontas de abdômen e antenas de machos com fêmeas, o perfil de expressão de cry2 apresentou uma maior diferença entre a amplitude da expressão no início da fase clara e o pico de expressão na fase escura, do que a diferença observada nos machos sozinhos. Já para os genes Orco e fruitless foram observados perfis arrítmicos. A ablação de antenas não afetou o perfil de expressão dos genes estudados, sugerindo que machos sem antenas conseguem perceber a presença de fêmeas por outros sentidos que não a audição e o olfato (ex: visão e tato). (AU)

Locomotor activity in males of Aedes aegypti can shift in response to females' presence.

BACKGROUND: The study of physiological and behavioral traits of mosquito vectors has been of growing relevance for the proposition of alternative methods for controlling vector-borne diseases. Despite this, most studies focus on the female's traits, including the behavior of host seeking, the physiology of disease transmission and the site-choice for oviposition. However, understanding the factors that lead to males' reproductive success is of utmost importance, since it can help building new strategies for constraining population growth. Male behavior towards mating varies widely among species and the communication between males and females is the first aspect securing a successful encounter. Here we used an automated monitoring system to study the profile of locomotor activity of Aedes aegypti males in response to female's presence in an adapted confinement tube. We propose a new method to quantify male response to the presence of females, which can be potentially tested as an indicator of the success of one male in recognizing a female for mating. RESULTS: Locomotor activity varies in daily cycles regulated by an endogenous clock and synchronized by external factors, such as light and temperature. Our results show the previously described startle response to light, which is displayed as a steep morning activity peak immediately when lights are on. Activity drops during the day and begins to rise again right before evening, happening about 1.5 h earlier in males than in females. Most interestingly, males' activity shows a double peak, and the second peak is very subtle when males are alone and relatively more pronounced when females are present in the confinement tubes. The switch in the peak of activity, measured by the herein suggested Peak Matching Index (PMI), was significantly different between males with and without females. CONCLUSIONS: The adapted monitoring system used here allowed us to quantify the response of individual males to nearby females in terms of the extent of the activity peak displacement. In this direction, we created the peak matching index (PMI), a new parameter that we anticipate could be interpreted as the inclination of males to respond to females' presence, and further tested as an indicator of the potential for finding females for mating.