optimizing-the-rearing-procedure-of-germ-free-wasps

Optimizing the Rearing Procedure of Germ-Free Wasps

Aseptic rearing technology is a method of culturing insects under sterile or almost sterile conditions, which can effectively eliminate the influence of ...

Temperate bacteriophage WO is a model system for studying tripartite interactions among viruses, bacteria, and eukaryotes, especially investigations of the genomic stability of obligate intracellular bacteria. Few WO genomes exist because of the difficulty in isolating viral DNA from eukaryotic hosts, and most reports are by-products of Wolbachia sequencing. Only one partial genome of a WO phage has been determined directly from isolated particles. We determine the complete genome sequence of prophage WO (WOSol) in Wolbachia strain wSol, which infects the fig wasp Ceratosolen solmsi (Hymenoptera: Chalcidoidea), by high-efficiency thermal asymmetric interlaced PCR. The genome of WOSol is highly degenerated and disrupted by a large region (14,267 bp) from Wolbachia. Consistent with previous molecular studies of multiple WO genomes, the genome of WOSol appears to have evolved by single nucleotide mutations and recombinations.

High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) for determination of a highly degenerated prophage WO genome in a Wolbachia strain infecting a fig wasp species.

Fig pollinating wasps form obligate symbioses with their fig hosts. This mutualism arose approximately 75 million years ago. Unlike many other intimate symbioses, which involve vertical transmission of symbionts to host offspring, female fig wasps fly great distances to transfer horizontally between hosts. In contrast, male wasps are wingless and cannot disperse. Symbionts that keep intimate contact with their hosts often show genome reduction, but it is not clear if the wide dispersal of female fig wasps will counteract this general tendency. We sequenced the genome of the fig wasp Ceratosolen solmsi to address this question.

Obligate mutualism within a host drives the extreme specialization of a fig wasp genome.

Cryptic prophages are genetically defective in their induction and propagation, and are simply regarded as genetic remnants. There are several putative cryptic WO prophages in the sequenced Wolbachia genomes. Whether they are lytic is unclear and their functions are poorly understood. Only three open reading frames (ORFs) in cryptic WO prophages have been reported to be actively transcribed.

Large proportion of genes in one cryptic WO prophage genome are actively and sex-specifically transcribed in a fig wasp species.

Wolbachia are widespread intracellular bacteria infecting the major classes of arthropods and some filarial nematodes. In arthropods, Wolbachia have evolved various intriguing reproductive manipulations, including cytoplasmic incompatibility, parthenogenesis, feminization, and male killing. Sixteen supergroups of Wolbachia have been identified, named A-Q (except G). Though Wolbachia present great diversity in arthropods, spiders, especially cave spiders, are still a poorly surveyed group of Wolbachia hosts. Here, we report a novel Wolbachia supergroup from nine Telema cave spiders (Araneae: Telemidae) based on five molecular markers (16S rRNA, ftsZ, gltA, groEL, and coxA). In addition, phage WO, which was previously reported only in Wolbachia supergroups A, B, and F, infects this new Wolbachia supergroup. We detected a 100% infection rate for phage WO and Wolbachia in Telema species. The phylogenetic trees of phage WO and Wolbachia are not congruent, which suggests that horizontal transfer of phage WO has occurred in these secluded species. Additionally, these data indicate Telema-Wolbachia-phage WO may be a good model for exploring the horizontal transfer history of WO among different host species.

Discovery of a new Wolbachia supergroup in cave spider species and the lateral transfer of phage WO among distant hosts.

Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control.

Combating mosquito-borne diseases using genetic control technologies.

A germ-free rearing system is a crucial method for host-microbiota interactions using Nasonia as a model system. The previous rearing media in 2012 introduced toxic factors like bleach and antibiotics, required significant effort and volume of media preparation, and the rearing protocols in 2012 and 2016 often resulted in embryos, larvae, and enclosing pupae drowning, underfed, or desiccating. In this work, we optimize the germ-free rearing media that excludes the toxic factors and provide a substrate for the developing animals to have constant access to media without the risk of drowning or desiccation. The new process resulted in an increase in full maturation of larvae to adults from 33 to 65%, with no effect on the rate of growth or final adult size. This significantly improves the applicability of germ-free rearing of Nasonia and potentially other parasitoids.

An optimized method for Nasonia germ-free rearing.

Mosquitoes bring global health problems by transmitting parasites and viruses such as malaria and dengue. Unfortunately, current insecticide-based control strategies are only moderately effective because of high cost and resistance. Thus, scalable, sustainable, and cost-effective strategies are needed for mosquito-borne disease control. Symbiont-based and genome engineering-based approaches provide new tools that show promise for meeting these criteria, enabling modification or suppression approaches. Symbiotic bacteria like Wolbachia are maternally inherited and manipulate mosquito host reproduction to enhance their vertical transmission. Genome engineering-based gene drive methods, in which mosquitoes are genetically altered to spread drive alleles throughout wild populations, are also proving to be a potentially powerful approach in the laboratory. Here, we review the latest developments in both symbionts and gene drive-based methods. We describe some notable similarities, as well as distinctions and obstacles, relating to these promising technologies.

Symbionts and gene drive: two strategies to combat vector-borne disease.

In recent years, with the development of microbial research technologies, microbiota research has received widespread attention. The parasitoid wasp genus Nasonia is a good model organism for studying insect behavior, development, evolutionary genetics, speciation, and symbiosis. This review describes key advances and progress in the field of the Nasonia-microbiome interactions. We provide an overview of the advantages of Nasonia as a model organism for microbiome studies, list research methods to study the Nasonia microbiome, and discuss recent discoveries in Nasonia microbiome research. This summary of the complexities of Nasonia-microbiome relationships will help to contribute to a better understanding of the interactions between animals and their microbiomes and establish a clear research direction for Nasonia-microbiome interactions in the future.

Nasonia-microbiome associations: a model for evolutionary hologenomics research.

Insects are one of the most important animal life forms on earth. Symbiotic microbes are closely related to the growth and development of the host insects and can affect pathogen transmission. For decades, various axenic insect-rearing systems have been developed, allowing further manipulation of symbiotic microbiota composition. Here we review the historical development of axenic rearing systems and the latest progress in using axenic and gnotobiotic approaches to study insect-microbe interactions. We also discuss the challenges of these emerging technologies, possible solutions to address these challenges, and future research directions that can contribute to a more comprehensive understanding of insect-microbe interactions.

Axenic and gnotobiotic insect technologies in research on host-microbiota interactions.

Mosquito-borne diseases like dengue and malaria cause a significant global health burden. Unfortunately, current insecticides and environmental control strategies aimed at the vectors of these diseases are only moderately effective in decreasing disease burden. Understanding and manipulating the interaction between the mosquito holobiont (i.e., mosquitoes and their resident microbiota) and the pathogens transmitted by these mosquitoes to humans and animals could help in developing new disease control strategies. Different microorganisms found in the mosquito's microbiota affect traits related to mosquito survival, development, and reproduction. Here, we review the physiological effects of essential microbes on their mosquito hosts; the interactions between the mosquito holobiont and mosquito-borne pathogen (MBP) infections, including microbiota-induced host immune activation and Wolbachia-mediated pathogen blocking (PB); and the effects of environmental factors and host regulation on the composition of the microbiota. Finally, we briefly overview future directions in holobiont studies, and how these may lead to new effective control strategies against mosquitoes and their transmitted diseases.

Article	Year
High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) for determination of a highly degenerated prophage WO genome in a Wolbachia strain infecting a fig wasp species. Applied and environmental microbiology\| PubMed ID: 24077701	2013
Obligate mutualism within a host drives the extreme specialization of a fig wasp genome. Genome biology\| PubMed ID: 24359812	2013
Large proportion of genes in one cryptic WO prophage genome are actively and sex-specifically transcribed in a fig wasp species. BMC genomics\| PubMed ID: 25311369	2014
Discovery of a new Wolbachia supergroup in cave spider species and the lateral transfer of phage WO among distant hosts. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases\| PubMed ID: 26997548	2016
Combating mosquito-borne diseases using genetic control technologies. Nature communications\| PubMed ID: 34282149	2021
An optimized method for Nasonia germ-free rearing. Scientific reports\| PubMed ID: 34997157	2022
Symbionts and gene drive: two strategies to combat vector-borne disease. Trends in genetics : TIG\| PubMed ID: 35314082	2022
Nasonia-microbiome associations: a model for evolutionary hologenomics research. Trends in parasitology\| PubMed ID: 36496327	2023
Axenic and gnotobiotic insect technologies in research on host-microbiota interactions. Trends in microbiology\| PubMed ID: 36906503	2023
Holobiont perspectives on tripartite interactions among microbiota, mosquitoes, and pathogens. The ISME journal\| PubMed ID: 37231184	2023

Guan-Hong Wang

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