Single-cell transcriptome maps of myeloid blood cell lineages in Drosophila.

The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution.

Genomic dissection of an extended phenotype: Oak galling by a cynipid gall wasp.

Galls are plant tissues whose development is induced by another organism for the inducer's benefit. 30,000 arthropod species induce galls, and in most cases the inducing effectors and target plant systems are unknown. Cynipid gall wasps are a speciose monophyletic radiation that induce structurally complex galls on oaks and other plants. We used a model system comprising the gall wasp Biorhiza pallida and the oak Quercus robur to characterise inducer and host plant gene expression at defined stages through the development of galled and ungalled plant tissues, and tested alternative hypotheses for the origin and type of galling effectors and plant metabolic pathways involved. Oak gene expression patterns diverged markedly during development of galled and normal buds. Young galls showed elevated expression of oak genes similar to legume root nodule Nod factor-induced early nodulin (ENOD) genes and developmental parallels with oak buds. In contrast, mature galls showed substantially different patterns of gene expression to mature leaves. While most oak transcripts could be functionally annotated, many gall wasp transcripts of interest were novel. We found no evidence in the gall wasp for involvement of third-party symbionts in gall induction, for effector delivery using virus-like-particles, or for gallwasp expression of genes coding for plant hormones. Many differentially and highly expressed genes in young larvae encoded secretory peptides, which we hypothesise are effector proteins exported to plant tissues. Specifically, we propose that host arabinogalactan proteins and gall wasp chitinases interact in young galls to generate a somatic embryogenesis-like process in oak tissues surrounding the gall wasp larvae. Gall wasp larvae also expressed genes encoding multiple plant cell wall degrading enzymes (PCWDEs). These have functional orthologues in other gall inducing cynipids but not in figitid parasitoid sister groups, suggesting that they may be evolutionary innovations associated with cynipid gall induction.

Elevated cytokine levels associated with acute kidney injury due to wasp sting.

This study mainly to explore the change of serum cytokines in wasp sting patients and the potential correlation between cytokines and acute kidney injury (AKI) due to wasp stings. The levels of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ in 33 wasp sting and 24 healthy people were measured by flow cytometry, the level of IL-17 was detected by enzyme-linked immunosorbent assay and the laboratory examination including inflammatory indicators, muscle enzyme markers, and renal function were detected by automatic biochemical analyzer, blood analyzer, and urine analyzer. The wasp sting patients were divided into AKI (n = 10) and non-AKI groups (n = 23). The correlation between the levels of serum cytokines and laboratory examination results was analyzed. The levels of IL-2, IL-6, IL-10, IFN-γ, and IL-17 were statistically increased in wasp sting patients compared with the controls (P < 0.05). IL-6, IL-10, and IL-17 levels were markedly increased in the AKI group compared with the non-AKI group (P < 0.05). Moreover, compared with non-AKI group, inflammatory markers and muscle enzyme markers were more abnormal in the AKI group. The positive rate of urinary occult blood in the AKI group was higher than that in the non-AKI group. The levels of IL-2, IL-4, IL-6, IFN-γ, and IL-17 correlated positively with white blood cell counts. The levels of IL-2, IL-4, IL-10, IFN-γ, and IL-17 correlated positively with the levels of serum creatinine. The levels of IL-2, IL-4, IL-10, IL-10, and IFN-γ correlated positively with the levels of C-reactive protein. The levels of IL-10, and IFN-γ correlated positively with urinary occult blood. Conclusion: Elevated levels of cytokines in wasp sting patients might be involved in the development and progression of acute kidney injury.

Mechanical stress to Drosophila larvae stimulates a cellular immune response through the JAK/STAT signaling pathway.

Acute inflammation can cause serious tissue damage and disease in physiologically-challenged organisms. The precise mechanisms leading to these detrimental effects remain to be determined. In this study, we utilize a reproducible means to induce cellular immune activity in Drosophila larvae in response to mechanical stress. That is, forceps squeeze-administered stress induces lamellocytes, a defensive hemocyte type that normally appears in response to wasp infestation of larvae. The posterior signaling center (PSC) is a cellular microenvironment in the larval hematopoietic lymph gland that is vital for lamellocyte induction upon parasitoid attack. However, we found the PSC was not required for mechanical stress-induced lamellocyte production. In addition, we observed that mechanical injury caused a systemic expression of Unpaired3. This cytokine is both necessary and sufficient to activate the cellular immune response to the imposed stress. These findings provide new insights into the communication between injured tissues and immune system induction, using stress-challenged Drosophila larvae as a tractable model system.

Larvae and Nests of Aculeate Hymenoptera (Hymenoptera: Aculeata) Nesting in Reed Galls Induced by Lipara spp. (Diptera: Chloropidae) with a Review of Species Recorded. Part II.

The ability of aculeate Hymenoptera to utilize wetlands is poorly understood, and descriptions of their nests and developmental stages are largely absent. Here we present results based on our survey of hymenopterans using galls induced by Lipara spp. flies on common reed Phragmites australis in the years 2015-2016. We studied 20,704 galls, of which 9,446 were longitudinally cut and the brood from them reared in the laboratory, while the remaining 11,258 galls reared in rearing bags also in laboratory conditions. We recorded eight species that were previously not known to nest in reed galls: cuckoo wasps Chrysis rutilans and Trichrysis pumilionis, solitary wasps Stenodynerus chevrieranus and Stenodynerus clypeopictus, and bees Pseudoanthidium tenellum, Stelis punctulatissima, Hylaeus communis and Hylaeus confusus. Forty five species of Hymenoptera: Aculeata are known to be associated with reed galls, of which 36 make their nests there, and the other are six parasitoids of the family Chrysididae and three cuckoo bees of the genus Stelis. Of these species, Pemphredon fabricii and in southern Europe also Heriades rubicola are very common in reed galls, followed by Hylaeus pectoralis and two species of the genus Trypoxylon. We also found new host-parasite associations: Chrysis angustula in nests of Pemphredon fabricii, Chrysis rutilans in nests of Stenodynerus clypeopictus, Trichrysis pumilionis in nests of Trypoxylon deceptorium, and Stelis breviuscula in nests of Heriades rubicola. We provide new descriptions of the nests of seven species nesting in reed galls and morphology of mature larvae of eight species nesting in reed galls and two parasitoids and one nest cleptoparasite. The larvae are usually very similar to those of related species but possess characteristics that make them easy to distinguish from related species. Our results show that common reeds are not only expansive and harmful, but very important for many insect species associated with habitats dominated by this plant species.

Screening and Analysis of Janelia FlyLight Project Enhancer-Gal4 Strains Identifies Multiple Gene Enhancers Active During Hematopoiesis in Normal and Wasp-Challenged Drosophila Larvae.

A GFP expression screen has been conducted on >1000 Janelia FlyLight Project enhancer-Gal4 lines to identify transcriptional enhancers active in the larval hematopoietic system. A total of 190 enhancers associated with 87 distinct genes showed activity in cells of the third instar larval lymph gland and hemolymph. That is, gene enhancers were active in cells of the lymph gland posterior signaling center (PSC), medullary zone (MZ), and/or cortical zone (CZ), while certain of the transcriptional control regions were active in circulating hemocytes. Phenotypic analyses were undertaken on 81 of these hematopoietic-expressed genes, with nine genes characterized in detail as to gain- and loss-of-function phenotypes in larval hematopoietic tissues and blood cells. These studies demonstrated the functional requirement of the cut gene for proper PSC niche formation, the hairy, Btk29A, and E2F1 genes for blood cell progenitor production in the MZ domain, and the longitudinals lacking, dFOXO, kayak, cap-n-collar, and delilah genes for lamellocyte induction and/or differentiation in response to parasitic wasp challenge and infestation of larvae. Together, these findings contribute substantial information to our knowledge of genes expressed during the larval stage of Drosophila hematopoiesis and newly identify multiple genes required for this developmental process.

Parasitoids of the eucalyptus gall wasp Leptocybe invasa (Hymenoptera: Eulophidae) in China.

Leptocybe invasa Fisher & La Salle (Hymenoptera, Eulophidae) is an invasive pest in Eucalyptus plantations throughout the world. Potential biological control agents for L. invasa were investigated in the Fujian, Guangdong, Hainan, Guangxi, Jiangxi, and Sichuan provinces of China, where Eucalyptus spp. have been severely damaged by the eucalyptus gall wasp. Three hymenopteran parasitoids of L. invasa were identified: Quadrastichus mendeli Kim & La Salle (Eulophidae), Aprostocetus causalis La Salle & Wu (Eulophidae), and Megastigmus viggianii Narendran & Sureshan (Torymidae); M. viggianii is newly recorded in China. The percentages of parasitization by Q. mendeli, A. causalis, and M. viggianii were 2.96%-19.53%, 2.30%-26.38%, and 24.93%, respectively. The longevity and body length of females were significantly greater than for males in A. causalis and M. viggianii. No males of Q. mendeli were found in China. These parasitoids could be used as biological agents for L. invasa in China.

Toxicity of Piper aduncum (Piperaceae) Essential Oil Against Euschistus heros (F.) (Hemiptera: Pentatomidae) and Non-Effect on Egg Parasitoids.

Plant essential oils have been recognized as significant natural resources for insecticides. Herein, we have assessed the toxicity of the essential oil of Piper aduncum (Piperaceae) against Euschistus heros (F.) (Hemiptera: Pentatomidae), a key soybean pest in Neotropical America. In addition, we have assessed its effect on the performance of egg parasitoids. The essential oil was obtained from the leaves of P. aduncum via hydrodistillation. Subsequently, bioassays of the concentration response to eggs (contact and immersion methods), nymphs, and adults (topical application) were conducted, to assess the lethal effects on the stink bug. We also evaluated the performance of parasitism and adult emergence of egg parasitoids, when the host eggs were treated with essential oil. In the egg bioassay, both exposure methods were efficient for unviable eggs (immersion LC50 = 15.64 mg mL-1; contact LC50 = 21.29 mg mL-1), with the highlight on the immersion method. The bioassay with nymphs indicated a higher toxicity of essential oil, with lower concentrations (LC50 = 11.37 mg mL-1) being required to cause the death of insects. For adults, a reduction in survival of insects was observed, and consequently, there was a reduction in the number of individuals in the next generation. Although the essential oil was toxic to E. heros, it exhibited lower toxicity for egg parasitoids, as there was no effect on parasitism and the emergence of wasps. We discuss likely explanations for such selectivity. In summary, we found that the essential oil was promising for the control of E. heros, because it caused deleterious effects at all development stages of the stink bug and had no effect on parasitism and emergence of the egg parasitoids, which suggested compatibility with biological control.

The bracovirus genome of the parasitoid wasp Cotesia congregata is amplified within 13 replication units, including sequences not packaged in the particles.

The relationship between parasitoid wasps and polydnaviruses constitutes one of the few known mutualisms between viruses and eukaryotes. Viral particles are injected with the wasp eggs into parasitized larvae, and the viral genes thus introduced are used to manipulate lepidopteran host physiology. The genome packaged in the particles is composed of 35 double-stranded DNA (dsDNA) circles produced in wasp ovaries by amplification of viral sequences from proviral segments integrated in tandem arrays in the wasp genome. These segments and their flanking regions within the genome of the wasp Cotesia congregata were recently isolated, allowing extensive mapping of amplified sequences. The bracovirus DNAs packaged in the particles were found to be amplified within more than 12 replication units. Strikingly, the nudiviral cluster, the genes of which encode particle structural components, was also amplified, although not encapsidated. Amplification of bracoviral sequences was shown to involve successive head-to-head and tail-to-tail concatemers, which was not expected given the nudiviral origin of bracoviruses.

Divergent host-plant use promotes reproductive isolation among cynipid gall wasp populations.

Ecological speciation occurs when reproductive isolation evolves as a consequence of divergent natural selection among environments. A direct prediction of this process is that ecologically divergent pairs of populations will exhibit greater reproductive isolation than ecologically similar pairs of populations. By comparing allopatric populations of the cynipid gall wasp Belonocnema treatae infesting Quercus virginiana and Quercus geminata, we tested the role that divergent host use plays in generating ecological divergence and sexual isolation. We found differences in body size and gall structure associated with divergent host use, but no difference in neutral genetic divergence between populations on the same or different host plant. We observed significant assortative mating between populations from alternative host plants but not between allopatric populations on the same host plant. Thus, we provide evidence that divergent host use promotes speciation among gall wasp populations.

The effects of parasite-derived immune-suppressive factors on the cellular innate immune and autoimmune responses of Drosophila melanogaster.

Immune-suppressive factors (ISFs) introduced into larvae of Drosophila melanogaster during infection by virulent endoparasitic wasps effectively block the innate immune response mediated by blood cells (hemocytes) but have little influence on the autoimmune response made by a tumor strain in which the blood cells manifest a similar response but instead target and destroy endogenous tissues. Quantitative hemocyte analyses indicate that ISFs interfere with the immune effector responses downstream of nonself recognition, hemocyte activation and differentiation, because these responses were manifested by tumor hosts, in which the parasitoids developed. The data suggest that once activated to encapsulate aberrant tissues, the target specificity of the autoimmune-activated hemocytes, and the genetic program underlying tumor formation, cannot be blocked by parasitoid-derived ISFs, which effectively inhibit identical hemocyte-mediated responses during parasitization.

Changes of haemolymph protein profile in the larva of Pericallia ricini (Fabricius) parasitised by the Braconid Wasp, Apanteles taragamae Viereck (Hymenoptera: Braconidae).

Parasitism by the braconid wasp, A. taragamae caused alterations in the haemolymph polypeptides of woolly bear larvae of P. ricini. Analysis of haemolymph proteins by SDS-PAGE and densitometry showed that the quantities of haemolymph proteins were reduced dramatically in the parasitised larvae. Simultaneously, parasitism induced large amount of 95 kDa polypeptides in the haemolymph of the parasitised larvae. Also, a remarkable induction of 43 and 45 kDa polypeptides which are not detectable in non-parasitised larvae appeared in the parasitised larvae.

Transformation of gypsy moth (Lymantria dispar) cell lines by infection with Glyptapanteles indiensis polydnavirus.

Glyptapanteles indiensis, a species of braconid parasitic wasp, infects its host Lymantria dispar (gypsy moth) with a polydnavirus (GiPDV) to suppress the host immune system during parasitization. Here it is shown that GiPDV can infect L. dispar cell lines and that a portion of the GiPDV genome is stably maintained in infected cells. Results of Southern hybridization analyses suggested that this portion of the GiPDV genome is integrated into the L. dispar cellular genome. This is the first report of an insect viral DNA molecule that can apparently integrate into lepidopteran insect cells.

The effects of parasitism by Glyptapanteles liparidis (Braconidae: Hymenoptera) on the hemolymph and total body composition of gypsy moth larvae (Lymantria dispar, Lymantriidae: Lepldoptera).

The hemolymph and total body tissue composition (carbohydrates, lipids, proteins, and free amino acids) of gypsy moth larvae (Lymantria dispar, Lymantriidae:Lepidoptera) were analyzed and compared with the composition of larvae infected by their main parasitoid, Glyptapanteles liparidis (Braconidae:Hymenoptera). In the body tissue the concentrations of total lipids and total proteins decreased, whereas the glycogen concentration in the total body tissue was significantly elevated and exceeded the concentration of the nonparasitized larvae by a factor of approximately 3. Trehalose, glucose, and sorbitol were found in the hemolymph of both parasitized and nonparasitized larvae, but levels of all three carbohydrates were significantly reduced in parasitized individuals. The total lipid concentration was not altered due to parasitism. In the hemolymph the concentrations of total proteins nearly doubled in parasitized larvae, whereas the total free amino acid concentration was reduced; however, the levels of some single amino acids were reduced and those of others were elevated in the parasitized larvae. The polypeptide profile of the host hemolymph provided one parasitism-specific polypeptide at approximately 80 kDa. The results demonstrate that parasitization of gypsy moths by G. liparidis leads to significant alterations in levels of nearly all substance classes within the host's hemolymph and tissue.

Structure of a growth-blocking peptide present in parasitized insect hemolymph.

Last instar larvae of the insect armyworm, Pseudaletia separata, parasitized with the parasitoid wasp, Apanteles kariyai, do not initiate metamorphosis and, ultimately, the wasp larvae emerge from the host larvae about 10 days after parasitization (Tanaka, T., Agui, N., and Hiruma, K. (1987) Gen. Comp. Endocrinol. 67, 364-374). It is necessary for the parasitoid wasp to perturb the armyworm's endocrinological processes that control normal metamorphosis from larvae to pupae. This endocrinological perturbation allows the parasitoid to complete its larval growth before emerging from the host larvae. It is obligatory for the parasitoid larvae to emerge while the host is still in a larval stage because the sclerotized pupal cuticle is impenetrable for the parasitoid larvae. A growth-blocking peptide with repressive activity against juvenile hormone esterase has been proven to exist in the parasitized host larval plasma (Hayakawa, Y. (1990) J. Biol. Chem. 265, 10813-10816). Here, I describe the detailed structure of this peptide and also the corresponding synthetic peptide to confirm this structure.