Alternative Splice in Alternative Lice.

Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation.

Corrigendum: Dichotomous sperm in Lepidopteran insects: a biorational target for pest management.

[This corrects the article DOI: 10.3389/finsc.2023.1198252.].

A sperm-activating trypsin-like protease from the male reproductive tract of Spodoptera litura: Proteomic identification, sequence characterization, gene expression profile, RNAi and the effects of ionizing radiation.

Like other lepidopteran insects, males of the tobacco cutworm moth, Spodoptera litura produce two kinds of spermatozoa, eupyrene (nucleate) and apyrene (anucleate) sperm. Formed in the testis, both kinds of sperm are released into the male reproductive tract in an immature form and are stored in the duplex region of the tract. Neither type of sperm is motile at this stage. When stored apyrene sperm from the duplex are treated in vitro with an extract of the prostatic region of the male tract, or with mammalian trypsin, they become motile; activation is greater and achieved more rapidly with increasing concentration of extract or enzyme. The activating effect of prostatic extract is blocked by soybean trypsin inhibitor (SBTI), also in a dose-dependent way. These results suggest that the normal sperm-activating process is due to an endogenous trypsin-like protease produced in the prostatic region. Proteomic analysis of S. litura prostatic extracts revealed a Trypsin-Like Serine Protease, TLSP, molecular weight 27 kDa, whose 199-residue amino acid sequence is identical to that of a predicted protein from the S. litura genome and is highly similar to predicted proteins encoded by genes in the genomes of several other noctuid moth species. Surprisingly, TLSP is only distantly related to Serine Protease 2 (initiatorin) of the silkmoth, Bombyx mori, the only identified lepidopteran protein so far shown to activate sperm. TLSP has features typical of secreted proteins, probably being synthesized as an inactive precursor zymogen, which is later activated by proteolytic cleavage. cDNA was synthesized from total RNA extracted from the prostatic region and was used to examine TLSP expression using qPCR. tlsp mRNA was expressed in both the prostatic region and the accessory glands of the male tract. Injection of TLSP-specific dsRNA into adult males caused a significant reduction after 24 h in tlsp mRNA levels in both locations. The number of eggs laid by females mated to adult males that were given TLSP dsRNA in 10 % honey solution, and the fertility (% hatched) of the eggs were reduced. Injecting pupae with TLSP dsRNA caused the later activation of apyrene sperm motility by adult male prostatic extracts to be significantly reduced compared to controls. Exposure of S. litura pupae to ionizing radiation significantly reduced expression of tlsp mRNA in the prostatic part and accessory gland of irradiated males in both the irradiated generation and also in their (unirradiated) F1 progeny. The implications of these findings for the use of the inherited sterility technique for the control of S. litura and other pest Lepidoptera are discussed.

Dichotomous sperm in Lepidopteran insects: a biorational target for pest management.

Lepidoptera are unusual in possessing two distinct kinds of sperm, regular nucleated (eupyrene) sperm and anucleate (apyrene) sperm ('parasperm'). Sperm of both types are transferred to the female and are required for male fertility. Apyrene sperm play 'helper' roles, assisting eupyrene sperm to gain access to unfertilized eggs and influencing the reproductive behavior of mated female moths. Sperm development and behavior are promising targets for environmentally safer, target-specific biorational control strategies in lepidopteran pest insects. Sperm dimorphism provides a wide window in which to manipulate sperm functionality and dynamics, thereby impairing the reproductive fitness of pest species. Opportunities to interfere with spermatozoa are available not only while sperm are still in the male (before copulation), but also in the female (after copulation, when sperm are still in the male-provided spermatophore, or during storage in the female's spermatheca). Biomolecular technologies like RNAi, miRNAs and CRISPR-Cas9 are promising strategies to achieve lepidopteran pest control by targeting genes directly or indirectly involved in dichotomous sperm production, function, or persistence.

Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts.

Current sequence databases now contain numerous whole genome sequences of pathogenic bacteria. However, many of the predicted genes lack any functional annotation. We describe an assumption-free approach, Rapid Virulence Annotation (RVA), for the high-throughput parallel screening of genomic libraries against four different taxa: insects, nematodes, amoeba, and mammalian macrophages. These hosts represent different aspects of both the vertebrate and invertebrate immune system. Here, we apply RVA to the emerging human pathogen Photorhabdus asymbiotica using "gain of toxicity" assays of recombinant Escherichia coli clones. We describe a wealth of potential virulence loci and attribute biological function to several putative genomic islands, which may then be further characterized using conventional molecular techniques. The application of RVA to other pathogen genomes promises to ascribe biological function to otherwise uncharacterized virulence genes.

Cuticular plasticization in the tick, Amblyomma hebraeum (Acari: Ixodidae): possible roles of monoamines and cuticular pH.

The degree of plasticization of the alloscutal cuticle of a 'hard' (ixodid) tick, Amblyomma hebraeum, and a 'soft' (argasid) tick, Ornithodoros moubata, was assessed throughout the blood-feeding period. Cuticle viscosity was calculated from rate of creep of cuticle under constant load using a Maxwell model. Feeding-related plasticization (i.e. increased rate of extension under a constant load) occurred in A. hebraeum but not in O. moubata. Maxwell viscosity of unfed A. hebraeum cuticle was relatively high (approximately 720 GPa s) but was significantly lower in feeding ticks. Small partially fed ticks displayed a viscosity of approximately 108 GPa s. Still lower values (42 GPa s) were observed in the largest of the engorged ticks. Following cessation of feeding, there was a significant but limited reversal in viscosity back to approximately 100 GPa s. The water content of cuticle of unfed A. hebraeum (23.4% of wet mass) rose sharply after the onset of feeding and reached a plateau value of 34.0% at a fed/unfed weight ratio of 3 and beyond. Ixodid ticks lay down new endocuticle during the feeding period. The observed increase in cuticle hydration suggests that both old and new cuticles are hydrated during feeding. Monoamines may play an important role in controlling cuticle viscosity. Dopamine (DA) injected into partially fed A. hebraeum caused plasticization. 5-Hydroxytryptamine (serotonin, 5-HT), which induces plasticization in the blood-sucking insect Rhodnius prolixus, had no statistically significant effect on tick cuticle. Octopamine (OA) and tyramine both caused cuticle stiffening (i.e. opposed plasticization). This suggests a possible inhibitory effect but co-injection of OA with DA did not reduce DA-induced plasticization. The mechanism leading to plasticization of tick cuticle may involve a change in cuticular pH. The viscosity of tick cuticle loops was highest at pH 8.0 (389 GPa s) and fell precipitously in the acidic range to a low value of 2.2 GPa s at pH 5.5-5.7. A cuticular pH of approximately 6.5 would account for the lowest viscosity observed under physiological conditions (42.4 GPa s for large, day 0, engorged ticks). The V-ATPase inhibitor, concanamycin A, was a potent inhibitor of DA-induced plasticization. These results are consistent with a model in which DA acts to cause plasticization through transport of H(+) ions into the cuticle. Measurement of cuticular ion (Na(+), K(+), Ca(2+), Mg(2+)) content did not suggest that plasticization is caused by any of these ions. Taken together, our results suggest that the mechanism of cuticular plasticization in feeding A. hebraeum is related to hydration, and involves the transport of H(+) ions into the sub-cuticular space by cells in the hypodermis. Feeding-induced plasticization was not observed in the rapid feeding tick, O. moubata.

The KdpD/KdpE two-component system of Photorhabdus asymbiotica promotes bacterial survival within M. sexta hemocytes.

Many bacteria persist within phagocytes, deploying complex sets of tightly regulated virulence factors to manipulate and survive within host cells. So far, no single factor has been identified that is sufficient to allow intracellular persistence of an otherwise non-pathogenic bacterium. Here we report that the two-component KdpD/KdpE sensor kinase/response regulator of the insect and human pathogen Photorhabdus asymbiotica (Pa) is sufficient to allow a harmless laboratory strain of E. coli to resist phagocytic killing and persist within insect hemocytes, ultimately killing the insect. Screening of a cosmid library of Pa in E. coli by injection into the moth Manduca sexta, previously identified three overlapping clones which caused the insect to cease feeding and subsequently die. Transposon mutagenesis revealed a cosmid encoded kdp high affinity potassium pump regulon was responsible for this phenotype. Gentamycin protection assays and confocal microscopy revealed the cosmid clones were persisting inside insect hemocytes far longer than control bacteria. Cloning and expression of PakdpD/kdpE alone into E. coli recapitulated the phenotype. Bioassay results and transcriptional analysis of various E. coli kdp mutants harboring the Pa kdp genes confirmed that Pa KdpD/KdpE was able to induce the E. coli kdp pump structural genes in response to exposure to insect hemocytes but not blood plasma alone. The finding that Pa KdpD/KdpE can facilitate resistance of E. coli to phagocytic killing suggests a central role for potassium in this process, supporting previous work implicating potassium sensing in virulence of other bacteria and also in the normal process of protease killing of engulfed bacteria by neutrophils.

A nematode symbiont sheds light on invertebrate immunity.

Photorhabdus bacteria live in a 'symbiosis of pathogens' with nematodes that invade and kill insects. Recent work has begun to use the power of the model insect Drosophila to dissect the molecular basis of the invertebrate immune response to the combined insult of the worms and their symbiotic bacterial pathogens. By using RNA interference, it is now also possible to dissect this complex tripartite interaction in a range of both model and non-model hosts.

RNAi suppression of recognition protein mediated immune responses in the tobacco hornworm Manduca sexta causes increased susceptibility to the insect pathogen Photorhabdus.

Bacterial pathogens either hide from or overcome the immune response of their hosts. Here we show that two different species of insect pathogenic bacteria, Photorhabdus luminescens TT01 and Photorhabdus asymbiotica ATCC43949, were both recognized by the immune system of their host Manduca sexta, as indicated by a rapid increase in the levels of mRNAs encoding three different inducible microbial recognition proteins, Hemolin, Immulectin-2 and peptidoglycan recognition protein. RNA interference (RNAi)-mediated inhibition of expression ("knock-down") of each of these genes at the level of both mRNA and protein was achieved through injection of double-stranded RNA (dsRNA). Knock-down of any one of these genes markedly decreased the ability of the insects to withstand infection when exposed to either species of Photorhabdus, as measured by the rate at which infected insects died. RNAi against Immulectin-2 caused the greatest reduction in host resistance to infection. The decreased resistance to infection was associated with reduced hemolymph phenoloxidase activity. These results show not only that Photorhabdus is recognized by the Manduca sexta immune system but also that the insect's immune system plays an active, but ultimately ineffective, role in countering infection.

Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference.

Prior infection of Manduca sexta caterpillars with the non-pathogenic bacterium Escherichia coli elicits effective immunity against subsequent infection by the usually lethal and highly virulent insect pathogen Photorhabdus luminescens TT01. Induction of this protective effect is associated with up-regulation of both microbial pattern recognition protein genes (hemolin, immulectin-2 and peptidoglycan recognition protein) and anti-bacterial effector genes (attacin, cecropin, lebocin, lysozyme and moricin). We used RNA interference to knock down over-transcription of members of both these sets of genes one at a time. Interfering with expression of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. RNAi knock-down of immulectin-2 caused the greatest reduction in immunity, followed by hemolin and peptidoglycan recognition protein (PGRP) in that order, to the extent that knock-down of any one of these three proteins left the insects more susceptible to P. luminescens infection than insects that had not experienced prior infection with E. coli. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. Knock-down of attacin, cecropin or moricin caused treated insects to be more susceptible to P. luminescens infection than controls that had been pre-infected with E. coli but which had not received the specific RNAi reagents, but they were still less susceptible than insects that had not been pre-infected with E. coli. RNAi knock-down with expression of lebocin or lysozyme had no effect on E. coli-induced immunity to P. luminescens, indicating that these effectors are not involved in the response. By bleeding pre-infected caterpillars and growing the pathogen directly within cell-free insect haemolymph, we showed that at least part of the protection elicited by previous exposure to E. coli is due to the presence of factors within the blood plasma that inhibit the growth of P. luminescens. The production of these factors is inhibited by RNAi treatment with ds-RNA reagents that knock down hemolin, immulectin-2, and PGRP. These results demonstrate that the insect immune system can be effectively primed by prior infection with non-pathogenic bacteria against subsequent infection by a highly virulent pathogen. Given the continuous normal exposure of insects to environmental and symbiotic bacteria, we suggest that prior infection is likely to play a significant and underestimated role in determining the level of insect immunity found in nature.

Microbial infection causes the appearance of hemocytes with extreme spreading ability in monolayers of the tobacco hornworm Manduca sexta.

The ability to adhere to and spread on a surface is a common property of insect blood cells. Spreading on a glass surface by insect hemocytes is often used as a measure of immune fitness that can be inhibited by some insect pathogens and parasites. Here, we report that upon infection of the tobacco hornworm Manduca sexta with either a fungus (Beauveria bassiana) or a bacterium (Photorhabdus luminescens), a new type of hemocyte, not previously observed in healthy insects, was found in hemocyte monolayers. These cells have a distinctive morphology, characterised by extreme spreading ability. They achieve a diameter of up to 120 microm after 1 h on glass coverslips and are therefore extremely thin. These hyper-spreading cells first appear in fungal-infected insects prior to hyphal growth. Their numbers later fall to zero as the pathogen begins to proliferate. The same hyper-spreading cells are induced after a 24 h delay following an injection of laminarin, a source of the fungal cell wall polymer beta-1,3-glucans. Wounding, on the other hand, did not cause the appearance of hyper-spreading cells. Evidence is presented here that is consistent with these spreading cells having a role in the cellular immune response of nodule formation.

Ocurrence of the antibiotic producing bacterium Burkholderia sp. in colonies of the leaf-cutting ant Atta sexdens rubropilosa.

Fungus garden material from recently established Atta sexdens rubropilosa colonies (6-12 months old) was sampled to detect antibiotic producing microorganisms that inhibited the growth of pathogens of insects and of the fungus gardens but did not affect their mutualistic fungus. A bacterium with activity against the entomopathogenic fungus Beauveria bassiana was isolated from 56% of the gardens tested (n=57) and identified from its biochemical profile and from 16S and 23S ribosomal DNA sequences as a member of the genus Burkholderia. The ant-associated Burkholderia isolates secreted a potent, anti-fungal agent that inhibited germination of conidia of the entomopathogenic fungi B. bassiana, Metarhizium anisopliae, of the saprophytic Verticillium lecanii, and also of a specialist fungus garden Escovopsis weberi. Growth of the ant's mutualist fungus was unaffected.

Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: evidence from Manduca sexta and Blattella germanica.

RNA interference (RNAi) is a specific gene silencing mechanism mediated by double-stranded RNA (dsRNA), which has been harnessed as a useful reverse genetics tool in insects. Unfortunately, however, this technology has been limited by the variable sensitivity of insect species to RNAi. We propose that rapid degradation of dsRNA in insect hemolymph could impede gene silencing by RNAi and experimentally investigate the dynamics of dsRNA persistence in two insects, the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. An ex vivo assay revealed that dsRNA was rapidly degraded by an enzyme in M. sexta hemolymph plasma, whilst dsRNA persisted much longer in B. germanica plasma. A quantitative reverse transcription PCR-based assay revealed that dsRNA, accordingly, disappeared rapidly from M. sexta hemolymph in vivo. The M. sexta dsRNAse is inactivated by exposure to high temperature and is inhibited by EDTA. These findings lead us to propose that the rate of persistence of dsRNA in insect hemolymph (mediated by the action of one or more nucleases) could be an important factor in determining the susceptibility of insect species to RNAi.

Induction of RNA interference genes by double-stranded RNA; implications for susceptibility to RNA interference.

Gene silencing by RNA interference (RNAi) can be a useful reverse genetics tool in eukaryotes. However, some species appear refractory to RNAi. To study the role of the differential expression of RNAi proteins in RNAi, we isolated partial dicer-2, argonaute-2 translin, vasa intronic gene (VIG) and tudor staphylococcus/micrococcal nuclease (TSN) genes from the tobacco hornworm, Manduca sexta, a well-studied insect model which we have found to be variably sensitive to RNAi. We found that the RNAi gene, translin, was expressed at minimal levels in M. sexta tissue and that there is a specific, dose-dependent upregulation of dicer-2 and argonaute-2 expression in response to injection with dsRNA, but no upregulation of the other genes tested. Upregulation of gene expression was rapid and transient. In order to prolong the upregulation we introduced multiple doses of dsRNA, resulting in multiple peaks of dicer-2 gene expression. Our results have implications for the design of RNAi experiments and may help to explain differences in the sensitivity of eukaryotic organisms to RNAi.

Insect immune responses to nematode parasites.

Host innate immunity plays a central role in detecting and eliminating microbial pathogenic infections in both vertebrate and invertebrate animals. Entomopathogenic or insect pathogenic nematodes are of particular importance for the control of insect pests and vectors of pathogens, while insect-borne nematodes cause serious diseases in humans. Recent work has begun to use the power of insect models to investigate host-nematode interactions and uncover host antiparasitic immune reactions. This review describes recent findings on innate immune evasion strategies of parasitic nematodes and host cellular and humoral responses to the infection. Such information can be used to model diseases caused by human parasitic nematodes and provide clues indicating directions for research into the interplay between vector insects and their invading tropical parasites.

Isolation and functional characterization of an allatotropin receptor from Manduca sexta.

Manduca sexta allatotropin (Manse-AT) is a multifunctional neuropeptide whose actions include the stimulation of juvenile hormone biosynthesis, myotropic stimulation, cardioacceleratory functions, and inhibition of active ion transport. Manse-AT is a member of a structurally related peptide family that is widely found in insects and also in other invertebrates. Its precise role depends on the insect species and developmental stage. In some lepidopteran insects including M. sexta, structurally-related AT-like (ATL) peptides can be derived from alternatively spliced mRNAs transcribed from the AT gene. We have isolated a cDNA for an AT receptor (ATR) from M. sexta by a PCR-based approach using the sequence of the ATR from Bombyx mori. The sequence of the M. sexta ATR is similar to several G protein-coupled receptors from other insect species and to the mammalian orexin receptor. We demonstrate that the M. sexta ATR expressed in vertebrate cell lines is activated in a dose-responsive manner by Manse-AT and each Manse-ATL peptide in the rank order ATL-I > ATL-II > ATL-III > AT, and functional analysis in multiple cell lines suggest that the receptor is coupled through elevated levels of Ca(2+) and cAMP. In feeding larvae, Manse-ATR mRNA is present at highest levels in the Malpighian tubules, followed by the midgut, hindgut, testes, and corpora allata, consistent with its action on multiple target tissues. In the adult corpora cardiaca--corpora allata complex, Manse-ATR mRNA is present at relatively low levels in both sexes.

Galleria mellonella as an infection model for Campylobacter jejuni virulence.

Larvae of Galleria mellonella (Greater Wax Moth) have been shown to be susceptible to Campylobacter jejuni infection and our study characterizes this infection model. Following infection with C. jejuni human isolates, bacteria were visible in the haemocoel and gut of challenged larvae, and there was extensive damage to the gut. Bacteria were found in the extracellular and cell-associated fraction in the haemocoel, and it was shown that C. jejuni can survive in insect cells. Finally, we have used the model to screen a further 67 C. jejuni isolates belonging to different MLST types. Isolates belonging to ST257 were the most virulent in the Galleria model, whereas those belonging to ST21 were the least virulent.

Dissecting the immune response to the entomopathogen Photorhabdus.

Bacterial pathogens either hide from or modulate the host's immune response to ensure their survival. Photorhabdus is a potent insect pathogenic bacterium that uses entomopathogenic nematodes as vectors in a system that represents a useful tool for probing the molecular basis of immunity. During the course of infection, Photorhabdus multiplies rapidly within the insect, producing a range of toxins that inhibit phagocytosis of the invading bacteria and eventually kill the insect host. Photorhabdus bacteria have recently been established as a tool for investigating immune recognition and defense mechanisms in model hosts such as Manduca and Drosophila. Such studies pave the way for investigations of gene interactions between pathogen virulence factors and host immune genes, which ultimately could lead to an understanding of how some Photorhabdus species have made the leap to becoming human pathogens.