Paolo Gabrieli

The Interaction between a Sexually Transferred Steroid Hormone and a Female Protein Regulates Oogenesis in the Malaria Mosquito Anopheles gambiae

Steroid hormones transferred by the male during sex trigger a molecular cascade of events that increases the reproductive success of females in Anopheles gambiae mosquitoes.

Sexual transfer of the steroid hormone 20E induces the postmating switch in Anopheles gambiae

Significance Anopheles gambiae females are the principal vectors of malaria, a disease that kills more than 600,000 people every year. Current control methods using insecticides to kill mosquitoes are threatened by the spread of resistance in natural populations. A promising alternative control strategy is based on interfering with mosquito reproduction to reduce the number of malaria-transmitting females. Here we show that a male hormone transferred to the female during sex induces large changes in female behavior. These changes, defined as the postmating switch, include a physical incapacity for fertilization by additional males and the ability to lay mature eggs. Tampering with the function of this hormone generates unprecedented opportunities to reduce the reproductive success of Anopheles mosquitoes and impact malaria transmission. Female insects generally mate multiple times during their lives. A notable exception is the female malaria mosquito Anopheles gambiae, which after sex loses her susceptibility to further copulation. Sex in this species also renders females competent to lay eggs developed after blood feeding. Despite intense research efforts, the identity of the molecular triggers that cause the postmating switch in females, inducing a permanent refractoriness to further mating and triggering egg-laying, remains elusive. Here we show that the male-transferred steroid hormone 20-hydroxyecdysone (20E) is a key regulator of monandry and oviposition in An. gambiae. When sexual transfer of 20E is impaired by partial inactivation of the hormone and inhibition of its biosynthesis in males, oviposition and refractoriness to further mating in the female are strongly reduced. Conversely, mimicking sexual delivery by injecting 20E into virgin females switches them to an artificial mated status, triggering egg-laying and reducing susceptibility to copulation. Sexual transfer of 20E appears to incapacitate females physically from receiving seminal fluids by a second male. Comparative analysis of microarray data from females after mating and after 20E treatment indicates that 20E-regulated molecular pathways likely are implicated in the postmating switch, including cytoskeleton and musculature-associated genes that may render the atrium impenetrable to additional mates. By revealing signals and pathways shaping key processes in the An. gambiae reproductive biology, our data offer new opportunities for the control of natural populations of malaria vectors.

Engineering the control of mosquito-borne infectious diseases

Recent advances in genetic engineering are bringing new promise for controlling mosquito populations that transmit deadly pathogens. Here we discuss past and current efforts to engineer mosquito strains that are refractory to disease transmission or are suitable for suppressing wild disease-transmitting populations.

Mating activates the heme peroxidase HPX15 in the sperm storage organ to ensure fertility in Anopheles gambiae

Significance Successful fertilization requires viable sperm and eggs to meet. Some insects, such as the Anopheles gambiae female mosquito, the principal vector of malaria, mate only once and keep sperm received from a male in a specialized sperm storage organ while eggs are developed after taking a blood meal. Sperm are kept functional for several weeks, but the factors and mechanisms that achieve this preservation are unknown in this mosquito. Here we identify a heme peroxidase HPX15 and other mechanisms activated by sex that are important to preserve the functionality of stored sperm and long-term fertility. Disrupting the reproductive cycle in field Anopheles would reduce numbers of mosquitoes transmitting malaria, aiding in the fight against one of the world’s deadliest diseases. Anopheles gambiae mosquitoes are major African vectors of malaria, a disease that kills more than 600,000 people every year. Given the spread of insecticide resistance in natural mosquito populations, alternative vector control strategies aimed at reducing the reproductive success of mosquitoes are being promoted. Unlike many other insects, An. gambiae females mate a single time in their lives and must use sperm stored in the sperm storage organ, the spermatheca, to fertilize a lifetimes supply of eggs. Maintenance of sperm viability during storage is therefore crucial to the reproductive capacity of these mosquitoes. However, to date, no information is available on the factors and mechanisms ensuring sperm functionality in the spermatheca. Here we identify cellular components and molecular mechanisms used by An. gambiae females to maximize their fertility. Pathways of energy metabolism, cellular transport, and oxidative stress are strongly regulated by mating in the spermatheca. We identify the mating-induced heme peroxidase (HPX) 15 as an important factor in long-term fertility, and demonstrate that its function is required during multiple gonotrophic cycles. We find that HPX15 induction is regulated by sexually transferred 20-hydroxy-ecdysone (20E), a steroid hormone that is produced by the male accessory glands and transferred during copulation, and that expression of this peroxidase is mediated via the 20E nuclear receptor. To our knowledge, our findings provide the first evidence of the mechanisms regulating fertility in Anopheles, and identify HPX15 as a target for vector control.

Transcriptome profiling of sexual maturation and mating in the Mediterranean fruit fly, Ceratitis capitata.

Sexual maturation and mating in insects are generally accompanied by major physiological and behavioural changes. Many of these changes are related to the need to locate a mate and subsequently, in the case of females, to switch from mate searching to oviposition behaviour. The prodigious reproductive capacity of the Mediterranean fruit fly, Ceratitis capitata, is one of the factors that has led to its success as an invasive pest species. To identify the molecular changes related to maturation and mating status in male and female medfly, a microarray-based gene expression approach was used to compare the head transcriptomes of sexually immature, mature virgin, and mated individuals. Attention was focused on the changes in abundance of transcripts related to reproduction, behaviour, sensory perception of chemical stimulus, and immune system processes. Broad transcriptional changes were recorded during female maturation, while post-mating transcriptional changes in females were, by contrast, modest. In male medfly, transcriptional changes were consistent both during maturation and as a consequence of mating. Of particular note was the lack of the mating-induced immune responses that have been recorded for Drosophila melanogaster, that may be due to the different reproductive strategies of these species. This study, in addition to increasing our understanding of the molecular machinery behind maturation and mating in the medfly, has identified important gene targets that might be useful in the future management of this pest.

Transcriptional profiles of mating-responsive genes from testes and male accessory glands of the Mediterranean fruit fly, Ceratitis capitata.

Background Insect seminal fluid is a complex mixture of proteins, carbohydrates and lipids, produced in the male reproductive tract. This seminal fluid is transferred together with the spermatozoa during mating and induces post-mating changes in the female. Molecular characterization of seminal fluid proteins in the Mediterranean fruit fly, Ceratitis capitata, is limited, although studies suggest that some of these proteins are biologically active. Methodology/Principal Findings We report on the functional annotation of 5914 high quality expressed sequence tags (ESTs) from the testes and male accessory glands, to identify transcripts encoding putative secreted peptides that might elicit post-mating responses in females. The ESTs were assembled into 3344 contigs, of which over 33% produced no hits against the nr database, and thus may represent novel or rapidly evolving sequences. Extraction of the coding sequences resulted in a total of 3371 putative peptides. The annotated dataset is available as a hyperlinked spreadsheet. Four hundred peptides were identified with putative secretory activity, including odorant binding proteins, protease inhibitor domain-containing peptides, antigen 5 proteins, mucins, and immunity-related sequences. Quantitative RT-PCR-based analyses of a subset of putative secretory protein-encoding transcripts from accessory glands indicated changes in their abundance after one or more copulations when compared to virgin males of the same age. These changes in abundance, particularly evident after the third mating, may be related to the requirement to replenish proteins to be transferred to the female. Conclusions/Significance We have developed the first large-scale dataset for novel studies on functions and processes associated with the reproductive biology of Ceratitis capitata. The identified genes may help study genome evolution, in light of the high adaptive potential of the medfly. In addition, studies of male recovery dynamics in terms of accessory gland gene expression profiles and correlated remating inhibition mechanisms may permit the improvement of pest management approaches.

Sniffing out chemosensory genes from the Mediterranean fruit fly, Ceratitis capitata.

The Mediterranean fruit fly, Ceratitis capitata (medfly), is an extremely invasive agricultural pest due to its extremely wide host range and its ability to adapt to a broad range of climatic conditions and habitats. Chemosensory behaviour plays an important role in many crucial stages in the life of this insect, such as the detection of pheromone cues during mate pursuit and odorants during host plant localisation. Thus, the analysis of the chemosensory gene repertoire is an important step for the interpretation of the biology of this species and consequently its invasive potential. Moreover, these genes may represent ideal targets for the development of novel, effective control methods and pest population monitoring systems. Expressed sequence tag libraries from C. capitata adult heads, embryos, male accessory glands and testes were screened for sequences encoding putative odorant binding proteins (OBPs). A total of seventeen putative OBP transcripts were identified, corresponding to 13 Classic, three Minus-C and one Plus-C subfamily OBPs. The tissue distributions of the OBP transcripts were assessed by RT-PCR and a subset of five genes with predicted proteins sharing high sequence similarities and close phylogenetic affinities to Drosophila melanogaster pheromone binding protein related proteins (PBPRPs) were characterised in greater detail. Real Time quantitative PCR was used to assess the effects of maturation, mating and time of day on the transcript abundances of the putative PBPRP genes in the principal olfactory organs, the antennae, in males and females. The results of the present study have facilitated the annotation of OBP genes in the recently released medfly genome sequence and represent a significant contribution to the characterisation of the medfly chemosensory repertoire. The identification of these medfly OBPs/PBPRPs permitted evolutionary and functional comparisons with homologous sequences from other tephritids of the genera Bactrocera and Rhagoletis.

Safe and fit genetically modified insects for pest control: from lab to field applications

Insect transgenesis is continuously being improved to increase the efficacy of population suppression and replacement strategies directed to the control of insect species of economic and sanitary interest. An essential prerequisite for the success of both pest control applications is that the fitness of the transformant individuals is not impaired, so that, once released in the field, they can efficiently compete with or even out-compete their wild-type counterparts for matings in order to reduce the population size, or to spread desirable genes into the target population. Recent research has shown that the production of fit and competitive transformants can now be achieved and that transgenes may not necessarily confer a fitness cost. In this article we review the most recent published results of the fitness assessment of different transgenic insect lines and underline the necessity to fulfill key requirements of ecological safety. Fitness evaluation studies performed in field cages and medium/large-scale rearing will validate the present encouraging laboratory results, giving an indication of the performance of the transgenic insect genotype after release in pest control programmes.

Function and composition of male accessory gland secretions in Anopheles gambiae: a comparison with other insect vectors of infectious diseases

Abstract Human malaria, a major public health burden in tropical and subtropical countries, is transmitted exclusively by the bite of a female Anopheles mosquito. Malaria control strategies aimed at inducing sexual sterility in natural vector populations are an attractive alternative to the use of insecticides. However, despite their importance as disease vectors, limited information is available on the molecular mechanisms regulating fertility in Anopheles mosquitoes. In the major malaria vector, An. gambiae, the full complement of sperm and seminal fluid required for a female’s lifelong egg production is obtained from a single mating event. This single mating has important consequences for the physiology and behavior of An. gambiae females: in particular, they become refractory to further insemination, and they start laying eggs. In other insects including Drosophila, similar post-copulatory changes are induced by seminal proteins secreted by the male accessory glands and transferred to the female during mating. In this review, we analyze the current state of knowledge on the function and characterization of male seminal proteins in An. gambiae, and provide a comparative assessment of the role of these male reproductive factors in other mosquito vectors of human disease in which female post-copulatory behavior has been studied. Knowledge of the factors and mechanisms regulating fertility in An. gambiae and other vectors can help the design of novel control strategies to fight the spread of disease.

Polyandry Is a Common Event in Wild Populations of the Tsetse Fly Glossina fuscipes fuscipes and May Impact Population Reduction Measures

Background Glossina fuscipes fuscipes is the main vector of human and animal trypanosomiasis in Africa, particularly in Uganda. Attempts to control/eradicate this species using biological methods require knowledge of its reproductive biology. An important aspect is the number of times a female mates in the wild as this influences the effective population size and may constitute a critical factor in determining the success of control methods. To date, polyandry in G.f. fuscipes has not been investigated in the laboratory or in the wild. Interest in assessing the presence of remating in Ugandan populations is driven by the fact that eradication of this species is at the planning stage in this country. Methodology/Principal Findings Two well established populations, Kabukanga in the West and Buvuma Island in Lake Victoria, were sampled to assess the presence and frequency of female remating. Six informative microsatellite loci were used to estimate the number of matings per female by genotyping sperm preserved in the female spermathecae. The direct count of the minimum number of males that transferred sperm to the spermathecae was compared to Maximum Likelihood and Bayesian probability estimates. The three estimates provided evidence that remating is common in the populations but the frequency is substantially different: 57% in Kabukanga and 33% in Buvuma. Conclusions/Significance The presence of remating, with females maintaining sperm from different mates, may constitute a critical factor in cases of re-infestation of cleared areas and/or of residual populations. Remating may enhance the reproductive potential of re-invading propagules in terms of their effective population size. We suggest that population age structure may influence remating frequency. Considering the seasonal demographic changes that this fly undergoes during the dry and wet seasons, control programmes based on SIT should release large numbers of sterile males, even in residual surviving target populations, in the dry season.