Deficiency of Brummer lipase disturbs lipid mobilization and locomotion, and impairs reproduction due to defects in the eggshell ultrastructure in the insect vector Rhodnius prolixus.

Rhodnius prolixus is a hematophagous insect, which feeds on large and infrequent blood meals, and is a vector of trypanosomatids that cause Chagas disease. After feeding, lipids derived from blood meal are stored in the fat body as triacylglycerol, which is recruited under conditions of energy demand by lipolysis, where the first step is catalyzed by the Brummer lipase (Bmm), whose orthologue in mammals is the adipose triglyceride lipase (ATGL). Here, we investigated the roles of Bmm in adult Rhodnius prolixus under starvation, and after feeding. Its gene (RhoprBmm) was expressed in all the analyzed insect organs, and its transcript levels in the fat body were not altered by nutritional status. RNAi-mediated knockdown of RhoprBmm caused triacylglycerol retention in the fat body during starvation, resulting in larger lipid droplets and lower ATP levels compared to control females. The silenced females showed decreased flight capacity and locomotor activity. When RhoprBmm knockdown occurred before the blood meal and the insects were fed, the females laid fewer eggs, which collapsed and showed low hatching rates. Their hemolymph had reduced diacylglycerol content and vitellogenin concentration. The chorion (eggshell) of their eggs had no difference in hydrocarbon amounts or in dityrosine crosslinking levels compared to control eggs. However, it showed ultrastructural defects. These results demonstrated that Bmm activity is important not only to guarantee lipid mobilization to maintain energy homeostasis during starvation, but also for the production of viable eggs after a blood meal, by somehow contributing to the right formation of the egg chorion.

Light and dark cycles modify the expression of clock genes in the ovaries of Aedes aegypti in a noncircadian manner.

Circadian oscillators (i.e., circadian clocks) are essential to producing the circadian rhythms observed in virtually all multicellular organisms. In arthropods, many rhythmic behaviors are generated by oscillations of the central pacemaker, specific groups of neurons of the protocerebrum in which the circadian oscillator molecular machinery is expressed and works; however, oscillators located in other tissues (i.e., peripheral clocks) could also contribute to certain rhythms, but are not well known in non-model organisms. Here, we investigated whether eight clock genes that likely constitute the Aedes aegypti clock are expressed in a circadian manner in the previtellogenic ovaries of this mosquito. Also, we asked if insemination by conspecific males would alter the expression profiles of these clock genes. We observed that the clock genes do not have a rhythmic expression profile in the ovaries of virgin (VF) or inseminated (IF) females, except for period, which showed a rhythmic expression profile in ovaries of IF kept in light and dark (LD) cycles, but not in constant darkness (DD). The mean expression of seven clock genes was affected by the insemination status (VF or IF) or the light condition (LD 12:12 or DD), among which five were affected solely by the light condition, one solely by the insemination status, and one by both factors. Our results suggest that a functional circadian clock is absent in the ovaries of A. aegypti. Still, their differential mean expression promoted by light conditions or insemination suggests roles other than circadian rhythms in this mosquito's ovaries.

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.

Identification of the sex chromosome system in a sand fly species, Lutzomyia longipalpis s.l.

In many animal species, sex determination is accomplished by heterogamety i.e., one of the sexes produces two types of gametes, which upon fertilization will direct the development toward males or females. Both male ("XY") and female ("ZW") heterogamety are known to occur and can be easily distinguished when the sex-chromosomes are morphologically different. However, this approach fails in cases of homomorphic sex chromosomes, such as the sand fly Lutzomyia longipalpis s.l. (Psychodidae, Diptera), which is the main vector of visceral leishmaniosis in Brazil. In order to identify the heterogametic sex in L. longipalpis s.l., we did a whole-genome sequencing of males and females separately and used the "Y chromosome Genome Scan" (YGS) method to find sex-specific sequences. Our results, which were confirmed by PCR, show that L. longipalpis s.l. has XY system. The YGS method can be especially useful in situations in which no morphological difference is observed in the sex-chromosomes or when fresh specimens are not readily available.

Silencing of ATG6 and ATG8 promotes increased levels of triacylglycerol (TAG) in the fat body during prolonged starvation periods in the Chagas disease vector Rhodnius prolixus.

Rhodnius prolixus is an obligatorily hematophagous insect known as an important vector of Chagas disease. Autophagy is a conserved cellular mechanism that acts in response to nutrient starvation, where components of the cytoplasm are sequestered by a double membrane organelle, named autophagosome, which is targeted to fuse with the lysosome for degradation. Lipophagy is the process of lipid degradation by selective autophagy, where autophagosomes sequester lipid droplets and degrade triacylglycerol (TAG) generating free fatty acids for ß-oxidation. Here, two essential genes of the autophagic pathway, Atg6/Beclin1 (RpAtg6) and Atg8/LC3 (RpAtg8), were silenced and the storage of lipids during starvation in Rhodnius prolixus was monitored. We found that RNAi knockdown of both RpAtg6 and RpAtg8 resulted in higher levels of TAG in the fat body and the flight muscle, 24 days after the blood meal, as well as a larger average diameter of the lipid droplets in the fat body, as seen by Nile Red staining under the confocal fluorescence microscope. Silenced starved insects had lower survival rates when compared to control insects. Accordingly, when examined during the starvation period for monitored activity, silenced insects had lower spontaneous locomotor activity and lower forced flight rates. Furthermore, we found that some genes involved in lipid metabolism had their expression levels altered in silenced insects, such as the Brummer lipase (down regulated) and the adipokinetic hormone receptor (up regulated), suggesting that, as previously observed in mammalian models, the autophagy and neutral lipolysis machineries are interconnected at the transcriptional level. Altogether, our data indicate that autophagy in the fat body is important to allow insects to mobilize energy from lipid stores.

Silencing of RpATG8 impairs the biogenesis of maternal autophagosomes in vitellogenic oocytes, but does not interrupt follicular atresia in the insect vector Rhodnius prolixus.

Follicular atresia is the mechanism by which the oocyte contents are degraded during oogenesis in response to stress conditions, allowing the energetic resources stored in the developing oocytes to be reallocated to optimize female fitness. Autophagy is a conserved intracellular degradation pathway where double-membrane vesicles are formed around target organelles leading to their degradation after lysosome fusion. The autophagy-related protein 8 (ATG8) is conjugated to the autophagic membrane and has a key role in the elongation and closure of the autophagosome. Here we identified one single isoform of ATG8 in the genome of the insect vector of Chagas Disease Rhodnius prolixus (RpATG8) and found that it is highly expressed in the ovary during vitellogenesis. Accordingly, autophagosomes were detected in the vitellogenic oocytes, as seen by immunoblotting and electron microscopy. To test if autophagosomes were important for follicular atresia, we silenced RpATG8 and elicited atresia in vitellogenic females by Zymosan-A injections. We found that silenced females were still able to trigger the same levels of follicle atresia, and that their atretic oocytes presented a characteristic morphology, with accumulated brown aggregates. Regardless of the difference in morphology, RpATG8-silenced atretic oocytes presented the same levels of protein, TAG and PolyP, as detected in control atretic oocytes, as well as the same levels of acidification of the yolk organelles. Because follicular atresia has the ultimate goal of restoring female fitness, we tested if RpATG8-silenced atresia would result in female physiology and behavior changes. Under insectarium conditions, we found that atresia-induced control and RpATG8-silenced females present no changes in blood meal digestion, survival, oviposition, TAG content in the fat body, haemolymph amino acid levels and overall locomotor activity. Altogether, we found that autophagosomes are formed during oogenesis and that the silencing of RpATG8 impairs autophagosome biogenesis in the oocytes. Nevertheless, regarding major macromolecule degradation and adaptations to the fitness costs imposed by triggering an immune response, we found that autophagic organelles are not essential for follicle atresia in R. prolixus.

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.

The influence of a light and dark cycle on the egg laying activity of Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae).

BACKGROUND: The epidemiological importance of the mosquito Aedes aegypti as a vector of multiple human pathogens has generated a growing number of studies on the physiology and behaviour of its blood-feeding females. The activity of oviposition is one of the critical elements contributing to the expansion of Ae. aegypti's populations. Although there is a vast literature about oviposition behaviour, significant specific knowledge about egg viability and female fertility under light and dark conditions is still lacking. OBJECTIVES: We studied, in controlled laboratory conditions, the effect that light and dark cycles have on the efficiency of oviposition by Ae. aegypti females. METHODS: Physiological assays were performed using synchronised eggs obtained from forced egg laying. The number and viability of eggs was analysed under three different light/dark regimes: LD12:12 (12 h of light and 12 h of dark), DD (constant darkness) and LL (constant light). FINDINGS AND CONCLUSIONS: Our results show that females prefer to lay their eggs in dark conditions, but maximising the number and viability of eggs requires the occurrence of a light/dark cycle. Ongoing research on this theme has the potential of contributing to the proposition of new strategies for control based on the failure of egg laying and hatching.

The influence of a light and dark cycle on the egg laying activity of Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae)

BACKGROUND The epidemiological importance of the mosquito Aedes aegypti as a vector of multiple human pathogens has generated a growing number of studies on the physiology and behaviour of its blood-feeding females. The activity of oviposition is one of the critical elements contributing to the expansion of Ae. aegypti's populations. Although there is a vast literature about oviposition behaviour, significant specific knowledge about egg viability and female fertility under light and dark conditions is still lacking. OBJECTIVES We studied, in controlled laboratory conditions, the effect that light and dark cycles have on the efficiency of oviposition by Ae. aegypti females. METHODS Physiological assays were performed using synchronised eggs obtained from forced egg laying. The number and viability of eggs was analysed under three different light/dark regimes: LD12:12 (12 h of light and 12 h of dark), DD (constant darkness) and LL (constant light). FINDINGS and CONCLUSIONS Our results show that females prefer to lay their eggs in dark conditions, but maximising the number and viability of eggs requires the occurrence of a light/dark cycle. Ongoing research on this theme has the potential of contributing to the proposition of new strategies for control based on the failure of egg laying and hatching.

Divergent abdominal bristle patterns in two distantly related drosophilids: antero-posterior variations and sexual dimorphism in a modular trait.

The number of neurosensory bristles on abdominal sternites of Drosophila is a most investigated trait for quantitative genetic studies. However, the developmental pattern expressed on successive segments in both sexes has remained so far a neglected field. We explored three aspects of this general problem with an isofemale line design: comparing two distantly related species, Drosophila melanogaster and Zaprionus indianus, investigating bristle number variation along the antero-posterior axis, and analysing the sexual dimorphism. Antero-posterior variations could be analysed from segment A2 to A7 in females, and A2-A5 in males. In D. melanogaster, males and females showed parallel changes with a consistently lower number in males. In Z. indianus females the number was quite stable along the abdomen, while in males an important antero-posterior increase was found. The sexual dimorphism was further analysed by considering the female-male correlation and the female/male ratio. The results suggest that sternite bristle number is determined by several developmental genetic systems. One is acting along the antero-posterior axis and may be associated to a gradient, since the genetic correlation decreases when more distant segments are compared. Another is acting in the same way on most segments of both sexes, since the female-male genetic correlation is similar between homologous and non-homologous segments. Finally, genes with specific sex effects are acting on A7 in females of both species, and on A5 in Z. indianus males. The overall architecture of female and male abdomen seems to be constrained by the development of reproductive organs. A large difference between species suggests, however, that the sexual dimorphism of abdominal bristle number is not evolutionarily constrained.

Sexual dimorphism of body size and sternopleural bristle number: a comparison of geographic populations of an invasive cosmopolitan drosophilid.

Zaprionus indianus is a cosmopolitan drosophilid, of Afrotropical origin, which has recently colonized South America. The sexual dimorphism (SD) of body size is low, males being almost as big as females. We investigated 10 natural populations, 5 from America and 5 from Africa, using the isofemale line technique. Three traits were measured on each fly: wing and thorax length and sternopleural bristle number. Two indices of SD were compared, and found to be highly correlated (r > 0.99). For the sake of simplicity, only the female/male (F/M) ratio was further considered. A significant genetic variability of SD was found in all cases, although with a low heritability (intra-class correlation of 0.13), about half the value found for the traits themselves. For size SD, we did not find any variation among continents or any latitudinal trend, and average values were 1.02 for wing length and 1.01 for thorax length. Bristle number SD was much greater (1.07). Among mass laboratory strains, SD was genetically much more variable than in recently collected populations, a likely consequence of laboratory drift. Altogether, SD, although genetically variable and prone to laboratory drift, is independent of size variations and presumably submitted to a stabilizing selection in nature.