Requirements for market entry of gene drive-modified mosquitoes for control of vector-borne diseases: analogies to other biologic and biotechnology products.

Gene drive-modified mosquitoes (GDMMs) are proposed as new tools for control and elimination of malaria and other mosquito-borne diseases, and promising results have been observed from testing conducted in containment. Although still at an early stage of development, it is important to begin now to consider approval procedures and market entry strategies for the eventual implementation of GDMMs in the context of disease control programs, as these could impact future research plans. It is expected that, as for other types of new products, those seeking to bring GDMMs to market will be required to provide sufficient information to allow the regulator(s) to determine whether the product is safe and effective for its proposed use.

Adult Anesthesia, Feeding, and Sex Separation.

The adult stage is the only nonaquatic stage of the mosquito. Both male and female mosquitoes require access to a source of sugar to survive. In the insectary, a temperature of ∼27°C and 80% relative humidity and a cycle of 12 h light:12 h dark light, ideally with a sunrise and sunset period, are necessary minimum conditions to mimic their natural environment.

Larval Rearing.

Mosquito larvae are aquatic and go through four development stages (larval instars L1-L4) before pupation. Species vary in the duration of larval development, and a variety of external factors affect the development rate (e.g.

Considerations for Rearing and Maintaining in the Laboratory.

mosquitoes can transmit several human pathogens, including viruses such as o'nyong-nyong and parasites including spp. and spp., which cause malaria and filariasis, respectively.

Egg Collection, Disinfection, and Hatching.

Gravid (i.e., with fully developed eggs), mated females typically lay their eggs directly on water ∼48-72 h after a blood meal.

Pupa Collection and Sex Identification.

For most species, larval-pupal metamorphosis commences ∼1 wk after egg hatching. However, depending on the amount of food provided, HO temperature, and larval density, the pupation process can be accelerated or delayed. Synchronous pupation is difficult to accomplish consistently, and, thus, pupae need to be separated from larvae daily.

Sterile Insect Technique (SIT) against Species Mosquitoes: A Roadmap and Good Practice Framework for Designing, Implementing and Evaluating Pilot Field Trials.

and are invasive mosquito species that impose a substantial risk to human health. To control the abundance and spread of these arboviral pathogen vectors, the sterile insect technique (SIT) is emerging as a powerful complement to most commonly-used approaches, in part, because this technique is ecologically benign, specific, and non-persistent in the environment if releases are stopped. Because SIT and other similar vector control strategies are becoming of increasing interest to many countries, we offer here a pragmatic and accessible 'roadmap' for the pre-pilot and pilot phases to guide any interested party.

Surviving the Journey: Comparisons of Temperature-Stabilizing Materials for Living Arthropod Shipments.

Shipments of living mosquitoes and other arthropods require temperatures that are within a range that is compatible with their health and survival. In addition to express shipping and insulated containers, shipments often include materials that either store heat (i.e.

Sterile Insect Technique: Lessons From the Past.

When E.F. Knipling conceived of the release of sexually sterile insects to suppress wild populations, he laid down several fundamental qualities that characterized suitable target species-some of which mosquitoes generally violate-including high reproductive rates and large population numbers.

Physiological responses to cryoprotectant treatment in an early larval stage of the malaria mosquito, Anopheles gambiae.

The development of cryopreservation protocols for Anopheles gambiae could significantly improve research and control efforts. Cryopreservation of any An. gambiae life stage has yet to be successful.

Trials of the Automated Particle Counter for laboratory rearing of mosquito larvae.

As a means of obtaining reproducible and accurate numbers of larvae for laboratory rearing, we tested a large-particle flow-cytometer type device called the 'Automated Particle Counter' (APC). The APC is a gravity-fed, self-contained unit that detects changes in light intensity caused by larvae passing the detector in a water stream and controls dispensing by stopping the flow when the desired number has been reached. We determined the accuracy (number dispensed compared to the target value) and precision (distribution of number dispensed) of dispensing at a variety of counting sensitivity thresholds and larva throughput rates (larvae per second) using < 1-day old Anopheles gambiae and Aedes aegypti larvae.

Anopheles gambiae (s.l.) is found where few are looking: assessing mosquito diversity and density outside inhabited areas using diverse sampling methods.

Background: One of the promising current approaches to curb malaria lies in genetic vector control, the implementation of which will require an improved understanding of the movement of genetic constructs among mosquito populations. To predict potential gene flow from one area to another, it is important to begin to understand mosquito dynamics outside of the commonly-sampled village areas, and thus how genes may move between villages. This study assessed the presence and relative abundance of mosquitoes in a 6-km corridor between two villages in western Burkina Faso.

Measuring and reducing biofilm in mosquito rearing containers.

Background: Mosquito rearing containers contain organic-rich water that nourishes numerous bacteria, some of which are capable of forming biofilms. Biofilm is broadly an extracellular polymeric matrix (EPS) in which living bacteria occur, and the accumulation of biofilm is possible during routine stock-keeping as most of these containers are re-used. Whether biofilm has an effect on the mosquito rearing is not a question that has been investigated, nor have measures to reduce biofilm in this context been systematically studied.

Fluorescent markers rhodamine B and uranine for Anopheles gambiae adults and matings.

Background: Marking mosquitoes is vital for mark-release-recapture and many laboratory studies, but their small size precludes the use of methods that are available for larger animals such as unique identifier tags and radio devices. Fluorescent dust is the most commonly used method to distinguish released individuals from the wild population. Numerous colours and combinations can be used, however, dust sometimes affects longevity and behaviour so alternatives that do not have these effects would contribute substantially.

Pragmatic selection of larval mosquito diets for insectary rearing of Anopheles gambiae and Aedes aegypti.

Larval mosquitoes are aquatic omnivorous scavengers which scrape food from submerged surfaces and collect suspended food particles with their mouth brushes. The composition of diets that have been used in insectaries varies widely though necessarily provides sufficient nutrition to allow colonies to be maintained. Issues such as cost, availability and experience influence which diet is selected.

Macrotrabecular Hepatocellular Carcinoma: An Aggressive Subtype of Hepatocellular Carcinoma.

The macrotrabecular (MT) pattern of hepatocellular carcinoma (HCC) has been suggested to represent a distinct HCC subtype. We retrospectively reviewed 231 HCC cases. Detailed pathologic evaluation for histologic patterns, including MT-pattern, was performed for each case and recorded as percentage involved at 10% intervals.

Downregulation of female doublesex expression by oral-mediated RNA interference reduces number and fitness of Anopheles gambiae adult females.

Background: Mosquito-borne diseases affect millions worldwide, with malaria alone killing over 400 thousand people per year and affecting hundreds of millions. To date, the best strategy to prevent the disease remains insecticide-based mosquito control. However, insecticide resistance as well as economic and social factors reduce the effectiveness of the current methodologies.

Seasonal malaria vector and transmission dynamics in western Burkina Faso.

Background: In the context of widespread mosquito resistance to currently available pesticides, novel, precise genetic vector control methods aimed at population suppression or trait replacement are a potentially powerful approach that could complement existing malaria elimination interventions. Such methods require knowledge of vector population composition, dynamics, behaviour and role in transmission. Here were characterized these parameters in three representative villages, Bana, Pala and Souroukoudingan, of the Sudano-Sahelian belt of Burkina Faso, a region where bed net campaigns have recently intensified.

Plasmodium falciparum (Haemosporodia: Plasmodiidae) and O'nyong-nyong Virus Development in a Transgenic Anopheles gambiae (Diptera: Culicidae) Strain.

Transgenic Anopheles gambiae Giles (Diptera: Culicidae) mosquitoes have been developed that confer sexual sterility on males that carry a transgene encoding a protein which cuts ribosomal DNA. A relevant risk concern with transgenic mosquitoes is that their capacity to transmit known pathogens could be greater than the unmodified form. In this study, the ability to develop two human pathogens in these transgenic mosquitoes carrying a homing endonuclease which is expressed in the testes was compared with its nontransgenic siblings.

Large-cage assessment of a transgenic sex-ratio distortion strain on populations of an African malaria vector.

Background: Novel transgenic mosquito control methods require progressively more realistic evaluation. The goal of this study was to determine the effect of a transgene that causes a male-bias sex ratio on Anopheles gambiae target populations in large insectary cages.

Methods: Life history characteristics of Anopheles gambiae wild type and Ag(PMB)1 (aka 124L-2) transgenic mosquitoes, whose progeny are 95% male, were measured in order to parameterize predictive population models.

Evolution of gene expression levels in the male reproductive organs of mosquitoes.

Modifications in gene expression determine many of the phenotypic differentiations between closely related species. This is particularly evident in reproductive tissues, where evolution of genes is more rapid, facilitating the appearance of distinct reproductive characteristics which may lead to species isolation and phenotypic variation. Large-scale, comparative analyses of transcript expression levels have been limited until recently by lack of inter-species data mining solutions.

Calcifying Nested Stromal-Epithelial Tumor of the Liver: An Update and Literature Review.

Calcifying nested stromal-epithelial tumor is a rare entity that has gone by a variety of names in the literature: ossifying malignant mixed epithelial and stromal tumor, ossifying stromal-epithelial tumor, and desmoplastic nested spindle cell tumor of the liver. To our knowledge, approximately 38 cases have been reported in the literature. The histogenesis is still largely unknown but histopathologically is characterized by nests of spindle and epithelioid cells in an organoid arrangement surrounded by a prominent dense myofibroblastic stroma with occasional psammomatous calcification and focal heterotopic ossification.

Gastric Adenocarcinoma of the Fundic Gland Type: Update and Literature Review.

Objectives: Gastric adenocarcinoma of the fundic gland type (GA-FG) is a newly described entity with a lack of awareness amongst general surgical pathologists and this review highlights the key features and controversies associated with this uncommon neoplasm.

Methods: A literature search through PubMed using synonyms for GA-FG was conducted to obtain 111 cases.

Results: GA-FG is a well-differentiated neoplasm of oxyntic mucosa, that is comprised of chief cells and parietal cells.