Infection Dynamics and Immune Response in a Newly Described Drosophila-Trypanosomatid Association

Trypanosomatid parasites are significant causes of human disease and are ubiquitous in insects. Despite the importance of Drosophila melanogaster as a model of infection and immunity and a long awareness that trypanosomatid infection is common in the genus, no trypanosomatid parasites naturally infecting Drosophila have been characterized. Here, we establish a new model of trypanosomatid infection in Drosophila-Jaenimonas drosophilae, gen. et sp. nov. As far as we are aware, this is the first Drosophila-parasitic trypanosomatid to be cultured and characterized. Through experimental infections, we find that Drosophila falleni, the natural host, is highly susceptible to infection, leading to a substantial decrease in host fecundity. J. drosophilae has a broad host range, readily infecting a number of Drosophila species, including D. melanogaster, with oral infection of D. melanogaster larvae resulting in the induction of numerous immune genes. When injected into adult hemolymph, J. drosophilae kills D. melanogaster, although interestingly, neither the Imd nor the Toll pathway is induced and Imd mutants do not show increased susceptibility to infection. In contrast, mutants deficient in drosocrystallin, a major component of the peritrophic matrix, are more severely infected during oral infection, suggesting that the peritrophic matrix plays an important role in mediating trypanosomatid infection in Drosophila. This work demonstrates that the J. drosophilae-Drosophila system can be a powerful model to uncover the effects of trypanosomatids in their insect hosts. IMPORTANCE Trypanosomatid parasites are ubiquitous in insects and are significant causes of disease when vectored to humans by blood-feeding insects. In recent decades, Drosophila has emerged as the predominant insect model of infection and immunity and is also known to be infected by trypanosomatids at high rates in the wild. Despite this, there has been almost no work on their trypanosomatid parasites, in part because Drosophila-specific trypanosomatids have been resistant to culturing. Here, we present the first isolation and detailed characterization of a trypanosomatid from Drosophila, finding that it represents a new genus and species, Jaenimonas drosophilae. Using this parasite, we conducted a series of experiments that revealed many of the unknown aspects of trypanosomatid infection in Drosophila, including host range, transmission biology, dynamics of infection, and host immune response. Taken together, this work establishes J. drosophilae as a powerful new opportunity to study trypanosomatid infections in insects

Neuroendocrine carcinoma arising in soft tissue: three case reports and literature review

<p>Abstract</p> <p>Background</p> <p>Neuroendocrine tumours (NET) are tumours arising from neuroendocrine cells of neural crest origin. They are characterised by the presence of neurosecretory granules which react positively to silver stains and to specific markers including neuron specific enolase, synaptophysin and chromogranin. Metastasis to the skin occurs infrequently but primary soft tissue NET is excessively rare.</p> <p>Case presentation</p> <p>We report our experience with 3 such cases. In the first case, the NET originated in muscle and was treated with wide surgical excision and adjuvant radiotherapy. The second case presented as a subcutaneous mass in the foot and the tumour was positive on ¹²³I mIBG scan. She has had prolonged recurrence-free survival following primary hypo-fractionated radiotherapy. In the third case, a cutaneous nodule proved to be a NET and at surgery, lymph node disease was present. He has remained disease-free after surgical excision without the need for external beam radiotherapy.</p> <p>Conclusion</p> <p>These tumours appear to have a good prognosis. Complete excision offers potentially curative treatment. Adjuvant radiotherapy may be helpful when the tumour margin is narrow. For patients with unresectable disease or where surgery would not be appropriate, radiotherapy appears to be an effective therapeutic option.</p

The role of defensive symbionts in host–parasite coevolution

Understanding the coevolution of hosts and parasites is a long‐standing goal of evolutionary biology. There is a well‐developed theoretical framework to describe the evolution of host–parasite interactions under the assumption of direct, two‐species interactions, which can result in arms race dynamics or sustained genotype fluctuations driven by negative frequency dependence (Red Queen dynamics). However, many hosts rely on symbionts for defence against parasites. Whilst the ubiquity of defensive symbionts and their potential importance for disease control are increasingly recognized, there is still a gap in our understanding of how symbionts mediate or possibly take part in host–parasite coevolution. Herein we address this question by synthesizing information already available from theoretical and empirical studies. First, we briefly introduce current hypotheses on how defensive mutualisms evolved from more parasitic relationships and highlight exciting new experimental evidence showing that this can occur very rapidly. We go on to show that defensive symbionts influence virtually all important determinants of coevolutionary dynamics, namely the variation in host resistance available to selection by parasites, the specificity of host resistance, and the trade‐off structure between host resistance and other components of fitness. In light of these findings, we turn to the limited theory and experiments available for such three‐species interactions to assess the role of defensive symbionts in host–parasite coevolution. Specifically, we discuss under which conditions the defensive symbiont may take over from the host the reciprocal adaptation with parasites and undergo its own selection dynamics, thereby altering or relaxing selection on the hosts' own immune defences. Finally, we address potential effects of defensive symbionts on the evolution of parasite virulence. This is an important problem for which there is no single, clear‐cut prediction. The selection on parasite virulence resulting from the presence of defensive symbionts in their hosts will depend on the underlying mechanism of defence. We identify the evolutionary predictions for different functional categories of symbiont‐conferred resistance and we evaluate the empirical literature for supporting evidence. We end this review with outstanding questions and promising avenues for future research to improve our understanding of symbiont‐mediated coevolution between hosts and parasites.ISSN:1469-185XISSN:1464-7931ISSN:0006-323

Data from: Generality of toxins in defensive symbiosis: ribosome-inactivating proteins and defense against parasitic wasps in Drosophila

While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. Here we test the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. We find strong evidence for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the α-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, we found that the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, escapes protection by Spiroplasma, and its ribosomes do not show high levels of depurination. We also show that fly ribosomes show little evidence of targeting by RIPs. Finally, we find that the genome of D. neotestacea’s defensive Spiroplasma encodes a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions

Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in <i>Drosophila</i>

<div><p>While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. <i>Spiroplasma</i> bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse <i>Drosophila</i> flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by <i>Spiroplasma</i>, and related to Shiga-like toxins in enterohemorrhagic <i>Escherichia coli</i>, in defense against a virulent parasitic nematode in the woodland fly, <i>Drosophila neotestacea</i>. Here we test the generality of RIP-mediated protection by examining whether <i>Spiroplasma</i> RIPs also play a role in wasp protection, in <i>D</i>. <i>melanogaster</i> and <i>D</i>. <i>neotestacea</i>. We find strong evidence for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, <i>Leptopilina heterotoma</i> and <i>Leptopilina boulardi</i>, exhibiting the hallmarks of RIP activity. In <i>Spiroplasma</i>-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the α-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, we found that the pupal ectoparasitic wasp, <i>Pachycrepoideus vindemmiae</i>, escapes protection by <i>Spiroplasma</i>, and its ribosomes do not show high levels of depurination. We also show that fly ribosomes show little evidence of targeting by RIPs. Finally, we find that the genome of <i>D</i>. <i>neotestacea’s</i> defensive <i>Spiroplasma</i> encodes a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.</p></div

The ectoparasitic wasp <i>Pachycrepoideus vindemmiae</i> successfully develops in <i>Spiroplasma</i>-positive <i>Drosophila melanogaster</i> and does not show evidence of RIP attack.

<p><i>Pachycrepoideus vindemmiae</i> (Pv) successfully parasitizes <i>Spiroplasma</i>-positive <i>D</i>. <i>melanogaster</i> (A). Neither fly nor wasp emergence was significantly affected by the presence of <i>Spiroplasma</i> (<i>p</i> ≥ .5). The proportion of depurinated ribosomes is much less than what is seen in <i>Leptopilina</i> species (<i>p</i> < .001; B). There is no difference in the level of intact <i>P</i>. <i>vindemmiae</i> ribosomes in <i>s</i>Mel-positive flies compared to levels in <i>s</i>Mel-negative flies (<i>p</i> = .778). Levels of depurinated ribosomes in <i>s</i>Mel-positive flies are modestly, albeit significantly, greater than in <i>s</i>Mel-negative flies (<i>p</i> = .004; C). Twelve <i>s</i>Mel-positive and nine <i>s</i>Mel-negative fly pupae were tested for RIP activity and jitter points are the mean of two technical replicates per wasp-infested larva. Significant comparisons from Tukey post hoc tests are labeled above boxplots.</p

Depurination of <i>Drosophila</i> ribosomes is minimal and does not explain host mortality during parasitic wasp infestation.

<p>Levels of intact <i>Drosophila</i> ribosomes are not significantly decreased in <i>Spiroplasma</i>-positive flies (blue), compared to <i>Spiroplasma</i>-negative flies (gold) (A,C,E; <i>p</i> = .348, <i>p</i> = .026, <i>p</i> = .693 for <i>L</i>. <i>heterotoma</i>-infested <i>D</i>. <i>melanogaster</i>, <i>L</i>. <i>boulardi</i>-infested <i>D</i>. <i>melanogaster</i> and <i>L</i>. <i>heterotoma</i>-infested <i>D</i>. <i>neotestacea</i>, respectively). Depurinated <i>Drosophila</i> ribosomes are significantly more abundant in <i>Spiroplasma</i>-positive (blue) compared to <i>Spiroplasma</i>-negative flies (gold; B,D,F; <i>p</i> < .001); however, there is no difference in abundance of depurinated <i>Drosophila</i> ribosomes between wasp-infested (blue) and uninfested hosts (pink) regardless of fly survival outcome following defense. Jitter points are the mean of two technical replicates per larva. (G) Levels of depurinated <i>Drosophila</i> ribosomes are plotted for samples of host hemolymph, hemocytes, bled larvae and unbled controls. Jitter points are the mean of two technical replicates per pool of eight larvae. The proportion of depurinated host and infesting wasp ribosomes from larval samples collected in a separate experiment are shown for comparison. Post hoc significance test results are shown above boxplots (Tukey tests).</p

Hallmark of RIP attack in wasps begins soon after hatching.

<p>Abundance of depurinated ribosomes across a timecourse of <i>L</i>. <i>heterotoma</i> infestation of <i>D</i>. <i>melanogaster</i> in the presence and absence of <i>s</i>Mel (n = 6 fly larvae or pupae per timepoint). Jitter points are the mean of two technical replicates per wasp-infested larva. Depurinated <i>L</i>. <i>heterotoma</i> ribosomes are detected 48 hours after oviposition in <i>s</i>Mel-positive <i>D</i>. <i>melanogaster</i>, with increasing levels at subsequent timepoints. For all significant comparisons, <i>p</i> < .001 (t-tests). Legends above boxplots indicate parasitic wasp developmental stage at each time point indicated.</p