Mosquitoes are the most notorious arthropod vectors of viral and parasitic diseases for which approximately half the world’s population, ~4,000,000,000, is at risk. Integrated pest management programs (IPMPs) have achieved some success in mitigating the regional transmission and persistence of these diseases. However, as many vector-borne diseases remain pervasive, it is obvious that IPMP successes have not been absolute in eradicating the threat imposed by mosquitoes. Moreover, the expanding mosquito geographic ranges caused by factors related to climate change and globalization (travel, trade, and migration), and the evolution of resistance to synthetic pesticides, present ongoing challenges to reducing or eliminating the local and global burden of these diseases, especially in economically and medically disadvantaged societies. Abatement strategies include the control of vector populations with synthetic pesticides and eco-friendly technologies. These “green” technologies include SIT, IIT, RIDL, CRISPR/Cas9 gene drive, and biological control that specifically targets the aquatic larval stages of mosquitoes. Regarding the latter, the most effective continues to be the widespread use of Lysinibacillus sphaericus (Ls) and Bacillus thuringiensis subsp. israelensis (Bti). Here, we present a review of the health issues elicited by vector mosquitoes, control strategies, and lastly, focus on the biology of Ls and Bti, with an emphasis on the latter, to which no resistance has been observed in the field.

The three most important commercial bacterial insecticides are all derived from subspecies of Bacillus thuringiensis (Bt). Specifically, Bt subsp. kurstaki (Btk) and Bt subsp. aizawai (Bta) are used to control larval lepidopteran pests. The third, Bt subsp. israelensis (Bti), is primarily used to control mosquito and blackfly larvae. All three subspecies produce a parasporal body (PB) during sporulation. The PB is composed of insecticidal proteins that damage the midgut epithelium, initiating a complex process that results in the death of the insect. Among these three subspecies of Bt, Bti is unique as it produces the most complex PB consisting of three compartments. Each compartment is bound by a multilaminar fibrous matrix (MFM). Two compartments contain one protein each, Cry11Aa1 and Cyt1Aa1, while the third contains two, Cry4Aa1/Cry4Ba1. Each compartment is packaged independently before coalescing into the mature spherical PB held together by additional layers of the MFM. This distinctive packaging process is unparalleled among known bacterial organelles, although the underlying molecular biology is yet to be determined. Here, we present structural and molecular evidence that the MFM has a hexagonal pattern to which Bti proteins Bt152 and Bt075 bind. Bt152 binds to a defined spot on the MFM during the development of each compartment, yet its function remains unknown. Bt075 appears to be derived from a bacteriophage major capsid protein (MCP), and though its sequence has markedly diverged, it shares striking 3-D structural similarity to the Escherichia coli phage HK97 Head 1 capsid protein. Both proteins are encoded on Bti’s pBtoxis plasmid. Additionally, we have also identified a six-amino acid motif that appears to be part of a novel molecular process responsible for targeting the Cry and Cyt proteins to their cytoplasmic compartments. This paper describes several previously unknown features of the Bti organelle, representing a first step to understanding the biology of a unique process of sorting and packaging of proteins into PBs. The insights from this research suggest a potential for future applications in nanotechnology.

Background: During development, Hand2 and Hoxd13 transcription factors (TFs) regulate Sonic hedgehog (Shh) expression in the zone of polarizing activity (ZPA) in the distal posterior limb mesoderm. The ZPA regulatory sequence (ZRS) is a conserved, limb-specific enhancer that controls Shh expression. The ZRS can be divided into 5′, central, and 3′ subdomains, each with an E-box site that can bind basic helix-loop-helix (bHLH) TFs like Hand2. In addition, two Hoxd13 sites are present in the 5′ and central subdomains. Hand2 purportedly binds the ZRS through the central E-box, and both Hand2 and Hoxd13 have been shown to activate the ZRS in vitro. We hypothesized that the central E-box was required for activity, while the other E-boxes and Hoxd13 sites localize ZRS activity to the distal posterior limb mesoderm.Methods: To identify the functional role of each subdomain, we generated three ZRS fragments (5′, central, and 3′) and combined fragment constructs to test subdomain collective contributions. Additionally, we disrupted the five binding sites, alone or in concert, using site-directed mutagenesis. All ZRS constructs were cloned into a GFP reporter and evaluated in an in vivo chicken limb bioassay. We validated our findings using select ZRS constructs in transgenic mice.Results: We found that the 3′ fragment was necessary for ZRS activity, while the 5′ and central fragments had no activity alone or when combined. However, combining the 3′ fragment with the 5′ fragment restored robust activity. Further, mutation of all five binding sites markedly reduced ZRS activity. Reinstating each of the Hoxd13 sites restored focal activity, while restoring the 5′ and central E-boxes had little effect. However, the 3′ E-box proved sufficient for robust activity even in the absence of the other four binding sites.Conclusion: Our data indicate that the ZRS 3′, not the central, subdomain is necessary for activity and contains the 3′ E-box that Hand2 likely uses to induce Shh expression, while the 5′ and central E-boxes appear to be inhibitory. Our data also suggest that the Hoxd13 binding sites promote localized activity within the ZPA.

Ascoviruses (AVs) are obligate intracellular pathogens that target the larval and pupal stages of lepidopteran insects, specifically moth caterpillars. AVs are unique among viruses in their (i) transmission mode, (ii) gross pathology, (iii) virion ultrastructure, (iv) genomic architecture featuring a remarkable combination of genes, and (v) ability to reprogram host cell death and lipid biosynthetic pathways to generate virion-containing vesicles (VCVs). The metabolically active acellular VCVs are repurposed to complete virogenesis and to facilitate dissemination by endoparasitoid wasps. Since their discovery in the late 20th century, research has focused on these distinctive traits and, to a lesser extent, their potential for biological control. Among AV proteins are the large DNA-binding P64 family, inhibitor of apoptosis-like proteins (IAPs), executioner caspase and caspase-like proteins, and lipid-modifying enzymes, which together drive their novel cytopathology. This review synthesizes current knowledge of AV biology and proposes a framework for understanding VCV formation by integrating predicted viral protein functions with host cellular pathways, including the ceramide–sphingosine-1-phosphate rheostat and apoptosis.

This review examines Bacillus thuringiensis subsp. israelensis (Bti) as both a highly selective microbial larvicide and a biological platform for protein storage and delivery, enabled by the structural features of its prokaryotic insect larvicidal organelle (PILO). Bti remains the most widely deployed biological agent for mosquito control. Decades of operational use demonstrate substantial public health benefits and only limited, manageable ecological tradeoffs within integrated vector management programs (IVMP). Its narrow host range underlies an excellent safety record for humans and other vertebrates. Moreover, laboratory and field studies consistently show that collateral effects are minimal, context dependent, reversible, and largely restricted to closely related non-target aquatic dipterans. These attributes have established Bti as a cornerstone of environmentally sustainable IVMP worldwide. Here, we synthesize current knowledge on Bti biology, ecological selectivity, field performance, and the resistance-management properties embedded in the molecular architecture of the PILO. Finally, we assess emerging opportunities and technical constraints in repurposing the PILO as an in vivo microbial factory for packaging heterologous proteins with potential pharmaceutical and industrial applications.

Bacillus thuringiensis subsp. israelensis (Bti) is the most widely used biological larvicide for mosquito control worldwide and a cornerstone of environmentally sustainable vector-management programs. Its long-term global deployment reflects a well-characterized balance between public-health benefit and manageable ecological tradeoffs within integrated vector management (IVM) frameworks. Bti combines high larvicidal efficacy, operational simplicity, and strong target specificity, resulting in an exceptional safety profile for humans and vertebrate wildlife. Decades of laboratory and field studies demonstrate that Bti is biologically selective rather than ecologically inert, with reproducible yet context-dependent effects confined to closely related non-target aquatic dipterans. This review links the molecular and toxicological foundations of Bti to its operational performance, ecological selectivity, resistance-mitigating properties, and sustained utility in mosquito-control programs. Beyond its established larvicidal function, Bti’s prokaryotic insect larvicidal organelle (PILO) represents an underexplored platform for heterologous intracellular protein assembly. Its dense packing, structural stability, and resistance to environmental and biochemical stress indicate an evolutionary specialization for high-capacity protein storage during sporulation. These properties support noncanonical applications in biomolecule storage and stabilization and motivate cautious exploration of environmentally responsive protein release strategies. Although significant mechanistic and translational challenges remain, particularly with respect to cargo trafficking, modularity, and purification, the architectural principles that have enabled effective mosquito control provide a strong foundation for extending PILO-based platforms beyond larvicidal applications.