Haobo Jiang

Extracellular serine protease (SP) cascades are central regulators of insect innate immunity. These cascades are negatively controlled by serine protease inhibitors (serpins) and fine-tuned by serine protease homologs (SPHs), which resemble SPs but lack catalytic activity. In Drosophila, a key SP cascade—the Toll-phenoloxidase (Toll-PO) pathway—governs both the melanization response and Toll-dependent antimicrobial peptide production. This cascade is triggered by secreted pattern-recognition receptors or microbial proteases and converges on 2 clip-domain SPs, Persephone and Hayan, which activate the Toll ligand Spätzle via the Spätzle-Processing Enzyme (SPE) and process prophenoloxidases. Here, we characterize the SPH Skanda and uncover its role in the Toll–PO cascade. skanda is genomically clustered with hayan and persephone and is transcriptionally induced upon infection. Skanda is unusual among SPHs, containing a long serine/threonine-rich region, 2 clip domains, and atypical disulfide bonds. Skanda is unstable and subject to cleavage by Grass. Functional assays show that Skanda dampens activation of Hayan, and to a lesser extent Persephone, within the Toll–PO cascade. Notably, skanda-deficient flies are highly susceptible to Staphylococcus aureus despite displaying normal Toll signaling and cuticular melanization. Moreover, compound mutants lacking 2 members of the hayan–psh–skanda cluster reveal a hidden contribution of Skanda to Toll activation in the absence of Persephone. Together, our results identify Skanda as a modulatory SPH that fine-tunes Toll pathway activity in concert with Persephone and Hayan.

Melanization is a central insect immune response to wounding and microbial invasion. It is mediated by a system of serine proteases and their homologs, mostly members of the CLIP subfamily, and leads to prophenoloxidase (proPO) activation and PO-mediated production of cytotoxic compounds and melanin polymers that kill and sequester invading pathogens. In mosquitoes, melanotic encapsulation provides an effective defense against malaria parasites, yet the composition and organization of the underlying serine protease system remain largely unclear. Guided by gene orthology and protein abundance, we expressed SP217, four CLIPCs, and 12 CLIPBs from Anopheles gambiae and reconstructed a five-step branched cascade using purified zymogens. SP217, an ortholog of Drosophila ModSP, initiates the cascade by activating proCLIPC1. CLIPC1 activates proCLIPB13 and proCLIPB47, two of the 10 orthologs of Drosophila Grass and Manduca HP5. CLIPB13 activates proCLIPC4, proCLIPC6, and proCLIPB10 whereas CLIPB47 activates proCLIPC6 only. In the next step, CLIPC6 activates proCLIPB4 and proCLIPB9. CLIPB9 and CLIPB10 function as proPO-activating proteases (PAPs), cleaving proPO2 to generate highly active PO2 in the presence of a cofactor. Together, these results have revealed a framework of the mosquito serine protease system that drives melanization. Our findings highlight both strong evolutionary conservation of the system among holometabolous insects and species-specific differences in its organization, providing a foundation for future analysis through combinational genetic perturbation.

Hemolymph coagulation is a vital component of insect immunity, rapidly preventing fluid loss and limiting pathogen spread after injury. In Manduca sexta larvae, paralytic peptide (PP) is a fast-acting cytokine activated by an unknown serine protease, and its influence on hemolymph mechanics remains largely unexplored. Here, we examine how PP exposure alters the extensional and surface rheological properties of larval hemolymph, providing insight into physical manifestation of the immune activation. Hemolymph collected from fifth-instar larvae exhibited weak viscoelastic behavior in 78% of samples, with a mean characteristic relaxation time of 2.2 ± 1.5 ms. Treatment with 10 μM PP resulted in a pronounced shift toward a more Newtonian-like behavior: only 54% of samples remained viscoelastic, and the mean relaxation time (1.0 ± 0.6 ms) was reduced by >2-fold. In samples subjected to repeated measurements, PP exposure caused a rapid loss of viscoelasticity on the timescale of seconds. Extensional rheology measurements were performed using hemolymph dripping and controlled electromagnetic pulling. The estimated shear rates and relaxation times were comparable, demonstrating that the experimental setup reproduces physiologically relevant extensional flow conditions associated with hemolymph dripping from the wound of caterpillars. Surface rheology was probed using magnetic rotational spectroscopy with magnetic microrods. No statistically significant differences in drag coefficients were detected between untreated and PP-treated hemolymph under Newtonian rotation regimes. However, frequent overdamped oscillations, transitions between rotation regimes, confined oscillation cascades, and reverse rotations were observed, revealing elastic responses not captured by a purely Newtonian model and consistent with Maxwell-type viscoelastic behavior. The absence of hemocytes at the droplet surface, together with these rheological signatures, suggests that PP-stimulated coagulation initiates within the bulk hemolymph and progresses outward, leaving the surface hemocyte-free. Overall, our findings demonstrate that PP rapidly and profoundly modulates hemolymph mechanics, inducing fast, bulk-driven coagulation that alters viscoelastic behavior on biologically relevant timescales.

The ReproSci project retrospectively analyzed the reproducibility of 1006 claims from 400 papers published between 1959 and 2011 in the field of Drosophila immunity. This project attempts to provide a comprehensive assessment, 14 years later, of the replicability of nearly all publications across an entire scientific community in experimental life sciences. We found that 61% of claims were verified, while only 7% were directly challenged (not reproducible), a replicability rate higher than previous assessments. Notably, 24% of claims had never been independently tested and remain unchallenged. We performed experimental validations of a selection of 45 unchallenged claim, that revealed that a significant fraction (38/45) of them is in fact non-reproducible. We also found that high-impact journals and top-ranked institutions are more likely to publish challenged claims. In line with the reproducibility crisis narrative, the rates of both challenged and unchallenged claims increased over time, especially as the field gained popularity. We characterized the uneven distribution of irreproducibility among first and last authors. Surprisingly, irreproducibility rates were similar between PhD students and postdocs, and did not decrease with experience or publication count. However, group leaders, who had prior experience as first authors in another Drosophila immunity team, had lower irreproducibility rates, underscoring the importance of early-career training. Finally, authors with a more exploratory, short-term engagement with the field exhibited slightly higher rates of challenged claims and a markedly higher proportion of unchallenged ones. This systematic, field-wide retrospective study offers meaningful insights into the ongoing discussion on reproducibility in experimental life sciences.

Drosophila immunity has been the focus of intense study and has impacted other research fields including innate immunity and agriculturally or epidemiologically relevant investigations of insect pests and vectors. Unsurprisingly for such a large body of work, some published results were later found to be irreproducible. Although some results have been contradicted in the literature, many have no published follow-up, either due to a lack of research or low motivation to publish negative or contradictory results. We have addressed this by performing a reproducibility project that analyses the verifiability of claims from articles published on Drosophila immunity before 2011. To assess reproducibility, we extracted claims from 400 articles on the Drosophila immune response to bacteria and fungi and performed preliminary verification by comparing these claims to other published literature in the field. Using alternative approaches, we also experimentally tested some ‘unchallenged’ claims, which had no published follow-up. The intent of this analysis was to centralize evidence on insights and findings to improve clarity for scientists that may base research programs on these data. All our data are published on a publicly available website associated with this article (https://ReproSci.epfl.ch/) that encourages community participation. This article provides a short summary of claims that were found to have contradictory evidence, which may help the community to assess past findings on Drosophila immunity and improve clarity going forward.

Extracellular serine protease (SP) cascades are central regulators of insect innate immunity. These cascades are negatively controlled by serine protease inhibitors (serpins) and fine-tuned by serine protease homologs (SPHs), which resemble SPs but lack catalytic activity. In Drosophila , a key SP cascade—the Toll–phenoloxidase (PO) pathway—governs both the melanization response and Toll-dependent antimicrobial peptide production. This cascade is triggered by secreted pattern-recognition receptors or microbial proteases and converges on two clip-domain SPs, Persephone and Hayan, which activate the Toll ligand Spätzle via the Spätzle-Processing Enzyme (SPE) and process prophenoloxidases. Here, we characterize the SPH Skanda and uncover its role in the Toll–PO cascade. skanda is genomically clustered with hayan and persephone and is transcriptionally induced upon infection. Skanda is unusual among SPHs, containing a long serine/threonine-rich region, two clip domains, and atypical disulfide bonds. Skanda is unstable and subject to cleavage by Grass. Functional assays show that Skanda dampens activation of Hayan, and to a lesser extent Persephone, within the Toll–PO cascade. Notably, skanda -deficient flies are highly susceptible to Staphylococcus aureus despite displaying normal Toll signaling and cuticular melanization. Moreover, compound mutants lacking two members of the hayan–psh–skanda cluster reveal a hidden contribution of Skanda to Toll activation in the absence of Persephone. Together, our results identify Skanda as a modulatory SPH that fine-tunes Toll pathway activity in concert with Persephone and Hayan.

Drosophila immunity has been the focus of intense study and has impacted other research fields including innate immunity and agriculturally or epidemiologically relevant investigations of insect pests and vectors. We have performed a reproducibility project that analyses the conceptual replicability of claims from articles published on Drosophila immunity before 2011. To assess replicability, we extracted claims from 400 articles on the Drosophila immune response to bacteria and fungi and performed preliminary verification by comparing these claims to other published literature in the field. Using alternative approaches, we also experimentally tested some ‘unchallenged’ claims, with no published follow-up. The intent of this analysis was to centralize evidence on insights and findings to improve clarity for scientists that may base research programs on these data. All our data are published on a publicly available website associated with this article ( https://ReproSci.epfl.ch/ ) that encourages community participation. This article provides a short summary of claims that were found to have contradictory evidence, which may help the community to assess past findings on Drosophila immunity and improve clarity going forward. Statistical analysis of this reproducibility study and metascience insights obtained from this approach are discussed in a companion article.

The ReproSci project retrospectively analyzed the conceptual replicability of 1006 claims from 400 papers published between 1959 and 2011 in the field of Drosophila immunity. This project represents an expert assessment of the replicability 14 years later, of the replicability of nearly all publications across an entire scientific community in experimental life sciences. We found that 61% of claims were verified, while only 7% were directly challenged (not replicable), a replicability rate higher than previous assessments. Notably, 24% of claims had never been independently tested and remain unchallenged. We performed experimental validations of a selection of 45 unchallenged claim, that revealed that a significant fraction (38/45) of them is in fact non-replicable. We also found that claims from top-ranked institutions were more likely to be challenged and that claims published in trophy journals showed a similar but weaker trend. In line with the reproducibility crisis narrative, the rates of both challenged and unchallenged claims increased over time, especially as the field gained popularity. We characterized the uneven distribution of irreplicability among first and last authors. Surprisingly, irreplicability rates were similar between PhD students and postdocs, and did not decrease with experience or publication count. However, group leaders, who had prior experience as first authors in another Drosophila immunity team, had lower irreplicability rates, underscoring the importance of early-career training. Finally, authors with a more exploratory, short-term engagement with the field exhibited slightly higher rates of challenged claims and a markedly higher proportion of unchallenged ones. While limited by its reliance on published literature, this systematic, field-wide retrospective study offers meaningful perspective into the ongoing discussion on reproducibility in experimental life sciences.