Fig 1.
Transcripts of some antimicrobial peptides are upregulated in adult fly head following behavioral training that produces long-term memory.
(A) Top, schematic representation of male courtship suppression training and mRNA sequencing. Bottom, Venn diagram depicting gene expression changes after 1X (blue), and 3X (yellow) training compared to the mock trained group. The antimicrobial peptide genes that are up-regulated after 3X training are indicated in the box. (B) Top, schematic representation of appetitive associative memory paradigm and mRNA sequencing. Bottom, Venn diagram comparing up or down regulated genes 4 hour after training with sucrose (two independent experiments carried out a month apart) or L-sorbose, compared to the CS only control group (Experiment 1), or the untrained control (Experiment 2). Antimicrobial peptide genes that are changed in both sucrose groups but not in L-sorbose are indicated in the box. (C) Venn diagram comparing genes that are significantly up or down regulated after male courtship suppression conditioning, and appetitive associative training. (D) Heat map showing the expression of immune genes 1 hour or 4 hours after different training conditions. The antimicrobial family is specifically altered in adult head under various behavioral conditions. Att: Attacin, CecC: CecropinC, Dpt: DiptericinA, DptB: DiptericinB, Dro: Drosocin, GNBP-like3: Gram-Negative Binding Protein like3, Imd: Immune deficiency, Mtk: Metchnikowin.
Fig 2.
DptB is required for long-term memory.
(A) DptB expression measured by RT-qPCR after male courtship suppression paradigm: 1X training, 2X training, 3X training, and 3X training with decapitated females (left). DptB expression measured by RT-qPCR 1 hour or 4 hours after associative appetitive memory paradigm (right). The data are mean of three technical repeats. (B) Knocking out DptB significantly reduces long-term memory. Long-term memory (24 hours after training) and short-term memory (15 minutes after training) of DptB null flies. Long-term memory deficit of DptB null flies is rescued by a DptB genomic construct or expression of HA-tagged DptB in head fat body. The dots represent individual memory index of outliers. (C) Removal of DptB impairs long-term appetitive memory. The data are plotted as mean ± SEM. Statistical analysis was performed using unpaired two-tailed t-test and (*) P ≤ 0.05, (**) P≤0.01, (***) P≤0.001 and (ns) not significant.
Fig 3.
DptB is required in the head fat body for long-term memory.
(A) Memory index 24 hours after male courtship suppression paradigm of different antimicrobial peptide RNAis expressed in the head fat body. Only DptB RNAi flies show a significant reduction in male courtship suppression memory compared to the control groups. (B) Short-term male courtship suppression memory is unaffected by DptB knock down. (C) Expression of DptB only in head fat body is important for courtship suppression memory. Statistical analysis was performed using one-way ANOVA comparison and (**) P≤0.01, (***) P≤0.001 and (ns) not significant. DptB: DiptericinB, GNBP-like3: Gram-Negative Binding Protein like3.
Fig 4.
GNBP-like 3 is required in neurons for long-term memory.
(A) Memory index 24 hours after male courtship suppression paradigm of different antimicrobial peptide RNAis expressed in neurons. The expression of GNBP-like3 RNAi in neurons significantly impaired long-term memory. (B) Short-term male courtship suppression memory is unaffected by GNBP-like3 knock down in neurons. (C) Expression of GNBP-like3 only in neurons is important for courtship suppression memory. (D) Deletion of GNBP-like3 gene impairs the ability to form long-term courtship suppression memory. (E) 24 h memory of wild type and AMP deficient flies at different sucrose concentration as US. The data are plotted as mean ± SEM. Statistical analysis was performed using one-way ANOVA comparison and (**) P≤0.01, (***) P≤0.001 and (ns) not significant in A. Statistical analysis was performed using unpaired two-tailed t-test and (*) P ≤ 0.05, (**) P≤0.01, (***) P≤0.001 and (ns) not significant in B, C, D, and E.
Fig 5.
DptB is enriched in the head fat body and GNBP-like 3 in neurons.
(A) EGFP expression when driven under DptB-regulatory sequences. Immunostaining reveals EGFP expression is confined to the outer layer of the head, outside the central brain (marked by nc82 antibody), where the head fat body is located. Wildtype brain serves as a control for non-specific immunoreactivity of anti-EGFP antibodies. (B) GNBP-like3 is present in synaptosomes. Top. The HA-tag was introduced into the C-terminal end of GNBP-like3 by homologous recombination using Crispr-Cas9. Middle. Schematic depiction of synaptosome purification from adult fly heads. The P4 fraction, between 0.8M and 1.2M sucrose, is most enriched for synaptic protein. Bottom. Different fractions from the synaptosome purification were blotted for GNBP-like3. Wild type flies serve as a control for HA-antibody specificity.
Fig 6.
DptB and GNBP-like3 have antimicrobial activity.
(A) Growth of bacteria bearing GNBP-like3, DptB, Drosocin, GNBP1, and mCherry as a control, in two different culture conditions: the rich LB media (left), and synthetic media with L-arabinose to induce the expression of the constructs (right). Bacterial growth is represented by OD600 over time. (B) Left, schematic representation of the experimental design. Right, bacterial plates showing that the fraction containing DptB slows bacterial growth. The known antimicrobial peptide Drosocin is used as a positive control. DptB: DiptericinB, GNBP1: Gram-Negative Binding Protein 1, GNBP-like3: Gram-Negative Binding Protein like3.