Heterosis Increases Fertility, Fecundity and Survival of Laboratory-Produced F1 Hybrid Males of the Malaria Mosquito Anopheles coluzzii

The success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically-modified male mosquitoes critically depends on mating between laboratory-reared males and wild females. Unfortunately, mosquito colonization, laboratory rearing, and genetic manipulations can all negatively affect male competitiveness. Heterosis is commonly used to produce domestic animals with enhanced vigor and homogenous genetic background and could therefore potentially improve the mating performance of mass-reared male mosquitoes. Here, we produced enhanced hybrid males of the malaria mosquito Anopheles coluzzii by crossing two strains colonized 35+ and 8 years ago. We compared the amount of sperm and mating plug proteins they transferred to females, as well as their insemination rate, reproductive success and longevity under various experimental conditions. Across experiments widespread adaptations to laboratory mating were detected in the older strain. In large-group mating experiments, no overall hybrid advantage in insemination rates and the amount of sperm and accessory gland proteins transferred to females was detected. Despite higher sperm activity, hybrid males did not appear more fecund. However, individual-male mating and laboratory-swarm experiments revealed that hybrid males, while inseminating fewer females than older inbred males, were significantly more fertile, producing larger mating plugs and drastically increasing female fecundity. Heterotic males also showed increased longevity. These results validate the use of heterosis for creating hybrid males with improved fitness from long-established inbred laboratory strains. Therefore, this simple approach could facilitate disease control strategies based on male mosquito releases with important ultimate benefits to human health

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

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 lifetime’s 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

Analysis of Two Novel Midgut-Specific Promoters Driving Transgene Expression in Anopheles stephensi Mosquitoes

Background: Tissue-specific promoters controlling the expression of transgenes in Anopheles mosquitoes represent a valuable tool both for studying the interaction between these malaria vectors and the Plasmodium parasites they transmit and for novel malaria control strategies based on developing Plasmodium-refractory mosquitoes by expressing anti-parasitic genes. With this aim we have studied the promoter regions of two genes from the most important malaria vector, Anopheles gambiae, whose expression is strongly induced upon blood feeding. Results: We analysed the A. gambiae Antryp1 and G12 genes, which we have shown to be midgut-specific and maximally expressed at 24 hours post-bloodmeal (PBM). Antryp1, required for bloodmeal digestion, encodes one member of a family of 7 trypsin genes. The G12 gene, of unknown function, was previously identified in our laboratory in a screen for genes induced in response to a bloodmeal. We fused 1.1 kb of the upstream regions containing the putative promoter of these genes to reporter genes and transformed these into the Indian malaria vector A. stephensi to see if we could recapitulate the expression pattern of the endogenous genes. Both the Antryp1 and G12 upstream regions were able to drive femalepredominant, midgut-specific expression in transgenic mosquitoes. Expression of the Antryp1-driven reporter in transgenic A. stephensi lines was low, undetectable by northern blot analysis, and failed to fully match the induction kinetics of the endogenous Antryp1 gene in A. gambiae. This incomplete conservation of expression suggests either subtle differences i

Transgenic technologies to induce sterility

The last few years have witnessed a considerable expansion in the number of tools available to perform molecular and genetic studies on the genome of Anopheles mosquitoes, the vectors of human malaria. As a consequence, knowledge of aspects of the biology of mosquitoes, such as immunity, reproduction and behaviour, that are relevant to their ability to transmit disease is rapidly increasing, and could be translated into concrete benefits for malaria control strategies. Amongst the most important scientific advances, the development of transgenic technologies for Anopheles mosquitoes provides a crucial opportunity to improve current vector control measures or design novel ones. In particular, the use of genetic modification of the mosquito genome could provide for a more effective deployment of the sterile insect technique (SIT) against vector populations in the field. Currently, SIT relies on the release of radiation sterilized males, which compete with wild males for mating with wild females. The induction of sterility in males through the genetic manipulation of the mosquito genome, already achieved in a number of other insect species, could eliminate the need for radiation and increase the efficiency of SIT-based strategies. This paper provides an overview of the mechanisms already in use for inducing sterility by transgenesis in Drosophila and other insects, and speculates on possible ways to apply similar approaches to Anopheles mosquitoes

Transgenic technologies to induce sterility

The last few years have witnessed a considerable expansion in the number of tools available to perform molecular and genetic studies on the genome of Anopheles mosquitoes, the vectors of human malaria. As a consequence, knowledge of aspects of the biology of mosquitoes, such as immunity, reproduction and behaviour, that are relevant to their ability to transmit disease is rapidly increasing, and could be translated into concrete benefits for malaria control strategies. Amongst the most important scientific advances, the development of transgenic technologies for Anopheles mosquitoes provides a crucial opportunity to improve current vector control measures or design novel ones. In particular, the use of genetic modification of the mosquito genome could provide for a more effective deployment of the sterile insect technique (SIT) against vector populations in the field. Currently, SIT relies on the release of radiation sterilized males, which compete with wild males for mating with wild females. The induction of sterility in males through the genetic manipulation of the mosquito genome, already achieved in a number of other insect species, could eliminate the need for radiation and increase the efficiency of SIT-based strategies. This paper provides an overview of the mechanisms already in use for inducing sterility by transgenesis in Drosophila and other insects, and speculates on possible ways to apply similar approaches to Anopheles mosquitoes

The interaction between a sexually transferred steroid hormone and a female protein regulates oogenesis in the malaria mosquito anopheles gambiae

Molecular interactions between male and female factors during mating profoundly affect the reproductive behavior and physiology of female insects. In natural populations of the malaria mosquito Anopheles gambiae, blood-fed females direct nutritional resources towards oogenesis only when inseminated. Here we show that the mating-dependent pathway of egg development in these mosquitoes is regulated by the interaction between the steroid hormone 20-hydroxy-ecdysone (20E) transferred by males during copulation and a female Mating-Induced Stimulator of Oogenesis (MISO) protein. RNAi silencing of MISO abolishes the increase in oogenesis caused by mating in blood-fed females, causes a delay in oocyte development, and impairs the function of male-transferred 20E. Co-immunoprecipitation experiments show that MISO and 20E interact in the female reproductive tract. Moreover MISO expression after mating is induced by 20E via the Ecdysone Receptor, demonstrating a close cooperation between the two factors. Male-transferred 20E therefore acts as a mating signal that females translate into an increased investment in egg development via a MISO-dependent pathway. The identification of this male–female reproductive interaction offers novel opportunities for the control of mosquito populations that transmit malaria

Conceptual framework and rationale

The sterile insect technique (SIT) has been shown to be an effective and sustainable genetic approach to control populations of selected major pest insects, when part of area-wide integrated pest management (AW-IPM) programmes. The technique introduces genetic sterility in females of the target population in the field following their mating with released sterile males. This process results in population reduction or elimination via embryo lethality caused by dominant lethal mutations induced in sperm of the released males. In the past, several field trials have been carried out for mosquitoes with varying degrees of success. New technology and experience gained with other species of insect pests has encouraged a reassessment of the use of the sterility principle as part of integrated control of malaria vectors. Significant technical and logistic hurdles will need to be overcome to develop the technology and make it effective to suppress selected vector populations, and its application will probably be limited to specific ecological situations. Using sterile males to control mosquito vector populations can only be effective as part of an AW-IPM programme. The area-wide concept entails the targeting of the total mosquito population within a defined area. It requires, therefore, a thorough understanding of the target pest population biology especially as regards mating behaviour, population dynamics, dispersal and level of reproductive isolation. The key challenges for success are: 1) devising methods to monitor vector populations and measuring competitiveness of sterile males in the field, 2) designing mass rearing, sterilization and release strategies that maintain competitiveness of the sterile male mosquitoes, 3) developing methods to separate sexes in order to release only male mosquitoes and 4) adapting suppression measures and release rates to take into account the high reproductive rate of mosquitoes. Finally, success in area-wide implementation in the field can only be achieved if close attention is paid to political, socio-economic and environmental sensitivities and an efficient management organization is established taking into account the interests of all potential stakeholders of an AW-IPM programme

Interspecific Hybridization Yields Strategy for South Pacific Filariasis Vector Elimination

Lymphatic filariasis (LF) is a global health problem, with over 120 million people affected annually. The current LF elimination program is focused on administering anti-filarial drugs to the entire at-risk population via annual mass drug administration (MDA). While the MDA program is proving effective in many areas, other areas may require augmentative measures such as vector control. An example of the latter is provided by some regions of the South Pacific where Aedes polynesiensis is the primary vector. Here, we describe a novel vector control approach based upon naturally occurring Wolbachia bacterial infections. Wolbachia are endosymbiotic intracellular bacteria that cause a form of sterility known as cytoplasmic incompatibility. We show that introgression crosses with mosquitoes that are infected with a different Wolbachia type results in an A. polynesiensis strain (designated ‘CP’) that is incompatible with naturally infected mosquitoes. No difference in mating competitiveness is observed between CP males and wild type males in laboratory assays. The results support continued development of the strategy as a tool to improve public health

"I have got something positive out of this situation": psychological benefits of caregiving in relatives of young people with muscular dystrophy.

This paper focuses on the psychological benefits of caregiving in key relatives of patients with muscular dystrophies (MD), a group of rare diseases characterized by progressive weakness and restriction of the patient's functional abilities. We describe whether relatives perceived caregiving to be a positive experience and test whether relatives' perceptions vary in relation to their view of the patient as a valued person, the degree of involvement in care, and the level of support provided by social network and professionals. The study sample included 502 key relatives of patients aged 4-25 years, suffering from Duchenne, Becker, or limb-girdle MD, in treatment for at least 6 months to one of the eight participating centers, living with at least one relative aged 18-80 years. Of key relatives, 88 % stated that they had gotten something positive out of the situation, 96 % considered their patients to be sensitive, and 94 % viewed their patients as talented. Positive aspects of caregiving were more recognized by key relatives who were more convinced that the patient was sensitive and who perceived that they received higher level of professional help and psychological social support. These results suggest that most key relatives consider that their caregiving experience has had a positive impact on their lives, despite the practical difficulties of caring for patients with MD. Professionals should help relatives to identify the benefits of caregiving without denying its difficulties. Clinicians themselves should develop positive attitudes towards family involvement in the care of patients with long-term diseases