The Role of Mosquito Hemocytes in Viral Infections.

Insect hemocytes are the only immune cells that can mount a humoral and cellular immune response. Despite the critical involvement of hemocytes in immune responses against bacteria, fungi, and parasites in mosquitoes, our understanding of their antiviral potential is still limited. It has been shown that hemocytes express humoral factors such as TEP1, PPO, and certain antimicrobial peptides that are known to restrict viral infections. Insect hemocytes also harbor the major immune pathways, such as JAK/STAT, TOLL, IMD, and RNAi, which are critical for the control of viral infection. Recent research has indicated a role for hemocytes in the regulation of viral infection through RNA interference and autophagy; however, the specific mechanism by which this regulation occurs remains uncharacterized. Conversely, some studies have suggested that hemocytes act as agonists of arboviral infection because they lack basal lamina and circulate throughout the whole mosquito, likely facilitating viral dissemination to other tissues such as salivary glands. In addition, hemocytes produce arbovirus agonist factors such as lectins, which enhance viral infection. Here, we summarize our current understanding of hemocytes' involvement in viral infections.

Trypsin-like Inhibitor Domain (TIL)-Harboring Protein Is Essential for Aedes aegypti Reproduction.

Cysteine-rich trypsin inhibitor-like domain (TIL)-harboring proteins are broadly distributed in nature but remain understudied in vector mosquitoes. Here we have explored the biology of a TIL domain-containing protein of the arbovirus vector Aedes aegypti, cysteine-rich venom protein 379 (CRVP379). CRVP379 was previously shown to be essential for dengue virus infection in Ae. aegypti mosquitoes. Gene expression analysis showed CRVP379 to be highly expressed in pupal stages, male testes, and female ovaries. CRVP379 expression is also increased in the ovaries at 48 h post-blood feeding. We used CRISPR-Cas9 genome editing to generate two mutant lines of CRVP379 with mutations inside or outside the TIL domain. Female mosquitoes from both mutant lines showed severe defects in their reproductive capability; mutant females also showed differences in their follicular cell morphology. However, the CRVP379 line with a mutation outside the TIL domain did not affect male reproductive performance, suggesting that some CRVP379 residues may have sexually dimorphic functions. In contrast to previous reports, we did not observe a noticeable difference in dengue virus infection between the wild-type and any of the mutant lines. The importance of CRVP379 in Ae. aegypti reproductive biology makes it an interesting candidate for the development of Ae. aegypti population control methods.

Pleiotropic Odorant-Binding Proteins Promote Aedes aegypti Reproduction and Flavivirus Transmission.

Insect odorant-binding proteins (OBPs) are small soluble proteins that have been assigned roles in olfaction, but their other potential functions have not been extensively explored. Using CRISPR/Cas9-mediated disruption of Aedes aegypti Obp10 and Obp22, we demonstrate the pleiotropic contribution of these proteins to multiple processes that are essential for vectorial capacity. Mutant mosquitoes have impaired host-seeking and oviposition behavior, reproduction, and arbovirus transmission. Here, we show that Obp22 is linked to the male-determining sex locus (M) on chromosome 1 and is involved in male reproduction, likely by mediating the development of spermatozoa. Although OBP10 and OBP22 are not involved in flavivirus replication, abolition of these proteins significantly reduces transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. These results extend our current understanding of the role of insect OBPs in insect reproduction and transmission of human pathogens, making them essential determinants of vectorial capacity. IMPORTANCE Aedes aegypti is the major vector for many arthropod-borne viral diseases, such as dengue, Zika, and chikungunya viruses. Previous studies suggested that odorant-binding proteins (OBPs) may have diverse physiological functions beyond the olfactory system in mosquitoes; however, these hypothesized functions have not yet been demonstrated. Here, we have used CRISPR/Cas9-based genome editing to functionally delete (knock out) Obp10 and Obp22 in Aedes aegypti. We showed that disruption of Obp10 or Obp22 significantly impairs female and male reproductive capacity by adversely affecting blood feeding, oviposition, fecundity and fertility, and the development of spermatozoa. We also showed that disruption of Obp10 or Obp22 significantly reduces the transmission of dengue and Zika viruses through a mechanism affecting secretion of viral particles into the saliva. Thus, our study is not only significant in understanding the functions of OBPs in mosquito biology, but also shows that OBPs may represent potent flavivirus transmission-blocking targets. Our study is in this regard particularly timely and important from a translational and public health perspective.

Whole-Genome Sequence of the Novel Enterobacter Bacteriophage Arya with an Integrase Pseudogene, Isolated from the Gut of the Formosan Subterranean Termite.

We isolated and sequenced the novel Enterobacter bacteriophage Arya from termite gut. The genome showed synteny to lytic bacteriophage genomes; however, the genome encodes a truncated, putatively nonfunctional integrase pseudogene. Lysogeny-related genes were previously observed in certain lytic phages, but their role and evolution remain unclear.

Whole-Genome Sequence of the Novel Temperate Enterobacter Bacteriophage Tyrion, Isolated from the Gut of the Formosan Subterranean Termite.

We sequenced the genome of the novel Enterobacter bacteriophage Tyrion isolated from termite gut. The bacteriophage is temperate in nature and belongs to the Podoviridae family. Many predicted bacteriophage genes showed similarity to prophage regions of the genomes of Enterobacteriaceae bacteria. This is the second bacteriophage isolated from termite gut.

Assessment of genetically engineered Trabulsiella odontotermitis as a 'Trojan Horse' for paratransgenesis in termites.

BACKGROUND: The Formosan subterranean termite, Coptotermes formosanus is an invasive urban pest in the Southeastern USA. Paratransgenesis using a microbe expressed lytic peptide that targets the termite gut protozoa is currently being developed for the control of Formosan subterranean termites. In this study, we evaluated Trabulsiella odontotermitis, a termite-specific bacterium, for its potential to serve as a 'Trojan Horse' for expression of gene products in termite colonies. RESULTS: We engineered two strains of T. odontotermitis, one transformed with a constitutively expressed GFP plasmid and the other engineered at the chromosome with a Kanamycin resistant gene using a non- disruptive Tn7 transposon. Both strains were fed to termites from three different colonies. Fluorescent microscopy confirmed that T. odontotermitis expressed GFP in the gut and formed a biofilm in the termite hindgut. However, GFP producing bacteria could not be isolated from the termite gut after 2 weeks. The feeding experiment with the chromosomally engineered strain demonstrated that T. odontotermitis was maintained in the termite gut for at least 21 days, irrespective of the termite colony. The bacteria persisted in two termite colonies for at least 36 days post feeding. The experiment also confirmed the horizontal transfer of T. odontotermitis amongst nest mates. CONCLUSION: Overall, we conclude that T. odontotermitis can serve as a 'Trojan Horse' for spreading gene products in termite colonies. This study provided proof of concept and laid the foundation for the future development of genetically engineered termite gut bacteria for paratransgenesis based termite control.

Complete Genome Sequence of Citrobacter Phage CVT22 Isolated from the Gut of the Formosan Subterranean Termite, Coptotermes formosanus Shiraki.

The complete genome of bacteriophage CVT22 infecting Citrobacter sp. strain TM1552 is reported here. Both the bacteriophage and Citrobacter sp. TM1552 were isolated from the gut of the Formosan subterranean termite, Coptotermes formosanus. This is the first report of a genome sequence of a bacteriophage isolated from the termite gut.