Helicoverpa are important polyphagous agricultural insect pests and they have a worldwide distribution. In this study, we report the bacterial community structure in the midgut of fifth instar larvae of Helicoverpa armigera, a species prevalent in the India, China, South Asia, South East Asia, Southern & Eastern Africa and Australia. Using culturable techniques, we isolated and identified members of Bacillus firmus, Bacillus niabense, Paenibacillus jamilae, Cellulomonas variformis, Acinetobacter schindleri, Micrococcus yunnanesis, Enterobacter sp., and Enterococcus cassiliflavus in insect samples collected from host plants grown in different parts of India. Besides these the presence of Sphingomonas, Ralstonia, Delftia, Paracoccus and Bacteriodetes was determined by culture independent molecular analysis. We found that Enterobacter and Enterococcus were universally present in all our Helicoverpa samples collected from different crops and in different parts of India. The bacterial diversity varied greatly among insects that were from different host plants than those from the same host plant of different locations. This result suggested that the type of host plant greatly influences the midgut bacterial diversity of H. armigera, more than the location of the host plant. On further analyzing the leaf from which the larva was collected, it was found that the H. armigera midgut bacterial community was similar to that of the leaf phyllosphere. This finding indicates that the bacterial flora of the larval midgut is influenced by the leaf surface bacterial community of the crop on which it feeds. Additionally, we found that laboratory made media or the artificial diet is a poor bacterial source for these insects compared to a natural diet of crop plant.

There are eight lysozyme genes in the Anopheles gambiae genome. Transcripts of one of these genes, LYSC-1, increased in Anopheles gambiae cell line 4a3B by 24 h after exposure to heat-killed Micrococcus luteus. Lysozyme activity was also identified in conditioned media from the cell line from which the protein was purified to homogeneity using ion exchange and gel filtration. Mass spectrometric analysis of the purified protein showed 100% identity to lysozyme c-1. Purified lysozyme c-1 was tested against non-mosquito derived as well as culturable bacteria isolated from mosquito midguts. Lysozyme c-1 had negligible effects on the growth of most mosquito-derived bacteria in vitro but did inhibit the growth of M. luteus. Although Lys c-1 did not directly kill most bacteria, knockdown of LYSC-1 resulted in significant mortality in mosquitoes subjected to hemocoelic infections with Escherichia coli but not M. luteus thus suggesting that this protein plays an important role in antibacterial defense against selected bacteria.

Background Plasmodium requires an obligatory life stage in its mosquito host. The parasites encounter a number of insults while journeying through this host and have developed mechanisms to avoid host defenses. Lysozymes are a family of important antimicrobial immune effectors produced by mosquitoes in response to microbial challenge.Methodology/Principal FindingsA mosquito lysozyme was identified as a protective agonist for Plasmodium. Immunohistochemical analyses demonstrated that Anopheles gambiae lysozyme c-1 binds to oocysts of Plasmodium berghei and Plasmodium falciparum at 2 and 5 days after infection. Similar results were observed with Anopheles stephensi and P. falciparum, suggesting wide occurrence of this phenomenon across parasite and vector species. Lysozyme c-1 did not bind to cultured ookinetes nor did recombinant lysozyme c-1 affect ookinete viability. dsRNA-mediated silencing of LYSC-1 in Anopheles gambiae significantly reduced the intensity and the prevalence of Plasmodium berghei infection. We conclude that this host antibacterial protein directly interacts with and facilitates development of Plasmodium oocysts within the mosquito.Conclusions/SignificanceThis work identifies mosquito lysozyme c-1 as a positive mediator of Plasmodium development as its reduction reduces parasite load in the mosquito host. These findings improve our understanding of parasite development and provide a novel target to interrupt parasite transmission to human hosts.

We sequenced and characterized two novel invertebrate-type lysozymes from the mosquito Anopheles gambiae. Alignment and phylogenetic analysis of these and a number of related insect proteins identified through bioinformatics strategies showed a high degree of conservation of this protein family throughout the Class Insecta. Expression profiles were examined for the two mosquito genes through semiquantitative and real-time PCR analysis. Lys i-1 transcripts were found in adult females in the fat body and Malpighian tubules, whereas Lys i-2 was detected only in fat bodies. Blood-feeding resulted in significantly increased transcript abundance for both genes in the midguts. Neither gene was upregulated following bacterial challenge.

This study provides evidence that insect-killing bacteria produce fabclavines that can be used to deter mosquito feeding.