Intergenomic Arms Races: Detection of a Nuclear Rescue Gene of Male-Killing in a Ladybird

Many species of arthropod are infected by deleterious inherited micro-organisms. Typically these micro-organisms are inherited maternally. Consequently, some, particularly bacteria of the genus Wolbachia, employ a variety of strategies that favour female over male hosts. These strategies include feminisation, induction of parthenogenesis and male-killing. These strategies result in female biased sex ratios in host populations, which lead to selection for host factors that promote male production. In addition, the intra-genomic conflict produced by the difference in transmission of these cytoplasmic endosymbionts and nuclear factors will impose a pressure favouring nuclear factors that suppress the effects of the symbiont. During investigations of the diversity of male-killing bacteria in ladybirds (Coccinellidae), unexpected patterns of vertical transmission of a newly discovered male-killing taxon were observed in the ladybird Cheilomenes sexmaculata. Initial analysis suggested that the expression of the bacterial male-killing trait varies according to the male(s) a female has mated with. By swapping males between females, a male influence on the expression of the male-killing trait was confirmed. Experiments were then performed to determine the nature of the interaction. These studies showed that a single dominant allele, which rescues male progeny of infected females from the pathological effect of the male-killer, exists in this species. The gene shows typical Mendelian autosomal inheritance and is expressed irrespective of the parent from which it is inherited. Presence of the rescue gene in either parent does not significantly affect the inheritance of the symbiont. We conclude that C. sexmaculata is host to a male-killing γ-proteobacterium. Further, this beetle is polymorphic for a nuclear gene, the dominant allele of which rescues infected males from the pathogenic effects of the male-killing agent. These findings represent the first reported case of a nuclear suppressor of male-killing in a ladybird. They are considered in regard to sex ratio and intra-genomic conflict theories, and models of the evolutionary dynamics and distribution of inherited symbionts.     

Positive Selection and Horizontal Gene Transfer in the Genome of a Male-Killing Wolbachia

Wolbachia are a genus of widespread bacterial endosymbionts in which some strains can hijack or manipulate arthropod host reproduction. Male killing is one such manipulation in which these maternally transmitted bacteria benefit surviving daughters in part by removing competition with the sons for scarce resources. Despite previous findings of interesting genome features of microbial sex ratio distorters, the population genomics of male-killers remain largely uncharacterized. Here, we uncover several unique features of the genome and population genomics of four Arizonan populations of a male-killing Wolbachia strain, wInn, that infects mushroom-feeding Drosophila innubila. We first compared the wInn genome with other closely related Wolbachia genomes of Drosophila hosts in terms of genome content and confirm that the wInn genome is largely similar in overall gene content to the wMel strain infecting D. melanogaster. However, it also contains many unique genes and repetitive genetic elements that indicate lateral gene transfers between wInn and non-Drosophila eukaryotes. We also find that, in line with literature precedent, genes in the Wolbachia prophage and Octomom regions are under positive selection. Of all the genes under positive selection, many also show evidence of recent horizontal transfer among Wolbachia symbiont genomes. These dynamics of selection and horizontal gene transfer across the genomes of several Wolbachia strains and diverse host species may be important underlying factors in Wolbachia’s success as a male-killer of divergent host species.     

Two New Strains of Wolbachia Affecting Natural Avocado Thrips

Wolbachia is an obligate intracellular bacterium with a high frequency of infection and a continental distribution in arthropods and nematodes. This endosymbiont can induce various reproductive phenotypes in their hosts and has been previously found naturally in several pests including thrips (Thripidae). These insects cause physical fruit damage and economic losses in avocado. The presence of Wolbachia was evaluated for the first time in avocado thrips populations of Frankliniella sp. and Scirtothrips hansoni sp.n. from eastern Antioquia. DNA from adult thrips individuals was used to assess the detection of Wolbachia by amplifying a fragment (600 bp) of the Wolbachia major surface protein (wsp) gene. Results confirmed the presence of two new Wolbachia strains in these two thrips species, with a higher percentage of natural infection in S. hansoni sp.n. The first Wolbachia species was found in Frankliniella sp. and belongs to supergroup A and the second was detected in S. hansoni sp.n. and is part of supergroup B. Wolbachia was more frequently found in females (32.73%), and only found in one male. Analysis of phylogenetic relationships, suggests that the two new Wolbachia sequences (wFran: Frankliniella and wShan: Scirtothrips hansoni) detected here represent two new groups for this endosymbiont. The haplotype network shows the presence of two possible haplotypes for each strain. Future studies to evaluate the possible use of Wolbachia as a control agent in avocado thrips are necessary.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00951-5.     

Spiroplasma in Drosophila melanogaster Populations: Prevalence, Male-Killing, Molecular Identification, and No Association with Wolbachia

Spiroplasma endosymbionts are maternally transmitted bacteria that may kill infected sons resulting in the production of female-biased broods. The prevalence of male killers varies considerably both between and within species. Here, we evaluate the spatial and temporal status of male-killing and non-male-killing Spiroplasma infection in three Brazilian populations of Drosophila melanogaster, nearly a decade after the first occurrence report for this species. The incidence of the male-killing Spiroplasma ranged from close to 0 to 17.7?% (so far the highest estimate for a Drosophila species) with a suggestion of temporal decline in a population. We also found non-male-killing Spiroplasma coexisting in one population at lower prevalence (3-5?%), and we did not detect it in the other two. This may be taken as a suggestion of a spreading advantage conferred by the male-killing strategy. Sequencing two loci, we identified the phylogenetic position of Spiroplasma strains from the three localities, showing that all strains group closely in the poulsonii clade. Due to intensive sampling effort, we were able to test the association between Spiroplasma infections and another widespread endosymbiont, Wolbachia, whose prevalence ranged from 81.8 to 100?%. The prevalence of Wolbachia did not differ between Spiroplasma-infected and uninfected strains in our largest sample nor were the prevalences of the two endosymbionts associated across localities.     

Response to Contrast Objects of the Seven-Spot Ladybird Coccinella septempunctata (Coleoptera, Coccinellidae)

The goal of the work was to establish whether the seven-spot ladybird Coccinella septempunctata has a spatial constancy towards the shape of images. The beetles were presented with black figures on the white wall of the cylindrical arena. The beetles were walking with different orientation of the body relative to the force of gravity: in the horizontal plane, on a three-ray labyrinth or on the flat ring in the bottom of the arena; with an inclination of 90° on the cylindrical ring wall; upwards on the vertical labyrinth or upside down, under a thin wire labyrinth stretched over the arena. The beetles in the horizontal position did not discriminate from each other differently orientated images, except for two types of the figures: vertical or horizontal bands (the choice was 80:20%) and meanders orientated downwards and upwards (the choice was 56:44%). The bands oriented vertically towards the Earth were preferred by the beetles when observed from the inclination position, but not in the case of a vertical ascension. The meanders were not discriminated when observed from the upside down position. The ascending oblique bands were preferred over the descending ones. No ability to discriminate the shape of objects was found in the ladybirds, and, correspondingly, they have no spatial constancy. The discrimination of figures and ornaments has the simplest mechanism: detection of the movement (directional) of a contrast margin.     

Toward a Wolbachia Multilocus Sequence Typing System: Discrimination of Wolbachia Strains Present in Drosophila Species

Among the diverse maternally inherited symbionts in arthropods, Wolbachia are the most common and infect over 20% of all species. In a departure from traditional genotyping or phylogenetic methods relying on single Wolbachia genes, the present study represents an initial Multilocus Sequence Typing (MLST) analysis to discriminate closely related Wolbachia pipientis strains, and additional data on sequence diversity in Wolbachia. We report a new phylogenetic characterization of four genes (aspC, atpD, sucB, and pdhB), and provide an expanded analysis of markers described in previous studies (16S rDNA, ftsZ, groEL, dnaA, and gltA). MLST analysis of the bacterial strains present in 16 different Drosophila–Wolbachia associations detected four distinct clonal complexes that also corresponded to maximum-likelihood identified phylogenetic clades. Among the 16 associations analyzed, six could not be assigned to MLST clonal complexes and were also shown to be in conflict with relationships predicted by maximum-likelihood phylogenetic inferences. The results demonstrate the discriminatory power of MLST for identifying strains and clonal lineages of Wolbachia and provide a robust foundation for studying the ecology and evolution of this widespread endosymbiont.     

Effects of pirimicarb,buprofezin and pymetrozine on survival,development and reproduction of Coccinella undecimpunctata (Coleoptera: Coccinellidae)

The effects of pirimicarb (a neurotoxin), buprofezin (an insect growth regulator) and pymetrozine (an antifeedant) on Coccinella undecimpunctata were assessed by studying the survival and development of all immature stages and the survival and reproductive performance of adults. Insecticides were sprayed at doses recommended by the manufacturers for the control of aphids and/or whiteflies. None of the three insecticides had a significant effect on the survival of C. undecimpunctata eggs. When sprayed on larvae, buprofezin significantly reduced survival to adulthood to <33%, compared to >45% for the control and other insecticide treatments. Rates of adult survival, fecundity, and fertility, and the percentage of egg hatch, were not significantly different between control and insecticide treatments. Thus, larval stages were more susceptible to insecticides than were adults. In general, pirimicarb and pymetrozine had no adverse effects on immature or adult stages of C. undecimpunctata, and hence are suitable for IPM of sucking pests.     

Identification of Wolbachia Strains in Mosquito Disease Vectors

Wolbachia bacteria are common endosymbionts of insects, and some strains are known to protect their hosts against RNA viruses and other parasites. This has led to the suggestion that releasing Wolbachia-infected mosquitoes could prevent the transmission of arboviruses and other human parasites. We have identified Wolbachia in Kenyan populations of the yellow fever vector Aedes bromeliae and its relative Aedes metallicus, and in Mansonia uniformis and Mansonia africana, which are vectors of lymphatic filariasis. These Wolbachia strains cluster together on the bacterial phylogeny, and belong to bacterial clades that have recombined with other unrelated strains. These new Wolbachia strains may be affecting disease transmission rates of infected mosquito species, and could be transferred into other mosquito vectors as part of control programs.     

Genotype Characterization of the Bacterium Expressing the Male-Killing Trait in the Ladybird Beetle Adalia bipunctata with Specific Rickettsial Molecular Tools

Volume 61, no. 4, p. 1433, Results, line 22: "RsaI" should read "AluI." Figure 3 legend, lines 5 and 6: "RsaI-digested" should read "AluI-digested." [This corrects the article on p. 1431 in vol. 61.].     

The influence of some honeybee products as a diet substitute on the different stages of Coccinella undecimpunctata L. in Egypt

Several natural diets of honeybee products were tested for rearing Coccinella undecimpunctata L. under laboratory conditions. Two groups of tested adult and larval insect have been fed on the tested diets, the first group were fed on the tested honey bee products diets without adding aphids, while the second group fed on the same honeybee products with aphids added. The oviposition period in the first group was significantly shorter than control. The tested diets supplied with royal jelly + aphid and/or pollen grains + aphid caused the longest oviposition period. Diets of aphid alone and bee honey with aphid recorded the shortest oviposition period. Fecundity of ladybird was the highest in the tested diets with aphids than diets without aphid and/or aphid diet alone. The durations of larval and pupal stages were significantly different when compared with the diets with or without aphid and/or aphid diet alone. Further results indicated that the diets of royal jelly caused the shortest duration in larval and pupal stages.     

Exopolysaccharides of Two Cyanobacterial Strains from Roman Hypogea

The cytomorphology and the exopolysaccharides of two heterocystous cyanobacteria, Scytonema ocellatum CP8-2 and Fischerella maior NAV 10 bis, isolated from biofilms collected in two Roman hypogea, were investigated from a cytochemical, ultrastructural and biochemical point of view. Cytochemical stains and observation using light and transmission electron microscopy showed a bi-layered sheath in both cyanobacteria, with a different composition in sulphated and carboxylic groups. RP-HPLC of the extracted polysaccharides forming the mucous matrix showed the neutral sugars glucose and arabinose and the charged galacturonic acid as the main monosaccharides. Circular dichroism spectra recorded at varying pH and temperature showed that these polymers followed a random coil model and did not present a structured organised conformation at ambient temperature. The outcomes of this study increase the understanding of colonization and biodeterioration of important archaeological and cultural sites by cyanobacterial biofilms and the role of the exopolymeric substances involved.     

Two endosymbiotic bacteria, Wolbachia and Arsenophonus, in the brown planthopper Nilaparvata lugens

The brown planthopper Nilaparvata lugens harbors intracellular fungal yeast-like symbionts and endosymbiotic bacteria, with the latter mainly comprising Wolbachia and Arsenophonus. In this study, Wolbachia or Arsenophonus were detected in all 15 brown planthopper populations collected from China and Southeastern Asian countries. Furthermore, Polymerase Chain Reaction (PCR) analysis of the individuals in a population that was infected by both Wolbachia and Arsenophonus showed that each individual was infected by only one of the two symbiotic bacteria. Real-time quantitative PCR showed that both endosymbionts are mainly localized in the mycetocytes of the fat body. Reciprocal crosses between the Wolbachia ⁺ and Arsenophonus ⁺ brown planthopper populations showed that both bacteria were maternally transmitted. Our results showed that the brown planthopper populations are extensively infected by Wolbachia or Arsenophonus, and the two bacteria may be exclusive in each host individual. This finding might be helpful for further studies on the biological functions of the endosymbiotic bacteria and will deepen our understanding of the complicated symbiosis system in this host.     

两株多孔菌属担子菌菌丝体中的三萜成分

用组织分离法从采集于广东鼎湖山的2株多孔菌属担子菌子实体中分离得到2株菌丝体,并对菌丝体分别进行固体发酵培养,培养物经乙醇提取后,用柱层析分离得到4个化合物,用光谱法鉴定均为羊毛脂烷型三萜,它们分别是多孔菌酸C（polyporenic acid C）（1）、茯苓酸（pachymic acid）（2）、齿孔酸（eburicoic acid）（3）和硫磺菌酸（sulphurenic acid）（4）。其中化合物2为首次从多孔菌属中发现。     

2株瘤胃纤维降解细菌的分离鉴定

从日粮精粗比为3:7的小尾寒羊×蒙古羊杂交一代绵羊的瘤胃内容物中分离到2株严格厌氧细菌,一株球菌WQ-1,1株弧菌WH-2,二者对滤纸有很好的降解能力。通过酶活力试验测得WQ-1的滤纸酶活、羧甲基纤维素酶活和β-葡萄糖苷酶活分别为0.66,7.0 U/mL和15.3 U/mL;WH-2的滤纸酶活、羧甲基纤维素酶活和β-葡萄糖苷酶活分别为0.52,6.9 U/mL和17.2 U/mL。经形态学、生理生化反应、生态特性和遗传型的鉴定,WQ-1归类为瘤胃球菌属(Ruminococcus)的黄色瘤胃球菌(Ruminococcus flavefaciens)。WH-2归类为丁酸弧菌属(Butyrivibrio)的溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)。     

Colour-associated mating success in a polymorphic Ladybird Beetle, Harmonia axyridis

1. Body size is often an important character in mating success, but has been only infrequently mentioned in regard to colour polymorphism. In this study, mating success was investigated in a colour polymorphic Ladybird Beetle, Harmonia axyridis , with reference both to colour morph and to body size.
2. In the non-melanic males the mating individuals were significantly larger than solitary individuals, while in melanic males there was no significant difference.
3. The mating pattern was close to random mating with respect to colour morph and there was no significant deviation.
4. The results suggest both body size and colour morph affect the male mating success and males of different body size obtain mating advantage according to the colour morph. Colour polymorphism in this species is controlled by alleles on a single locus. Thus, the alleles on that locus significantly influence the effect of selection on the quantitative character.     

Amino Acid Differences in a 30S Ribosomal Protein from Two Strains of Escherichia coli

The 30S ribosomal proteins of the K-12 and B strains of Escherichia coli differ in at least one protein component. This component, which is allelic in the two strains, has been isolated from both organisms. Amino acid analyses show that the protein from strain B contains between 20 and 28 more amino acids than does the analogue protein from strain K-12.     

Behavior of Wolbachia endosymbionts from Drosophila simulans in Drosophila serrata, a novel host

Many species harbor the incompatibility-inducing microbe Wolbachia, a maternally inherited endoparasite that causes reduced egg hatch in crosses between infected males and uninfected females. Infected females are immune to this effect, which gives them a relative fitness advantage that results in the spread of the infection. The strength of incompatibility, fitness deficits associated with the infection, and transmission rate from mother to offspring largely determine the rate and extent of spread of Wolbachia in a population. We transferred Wolbachia from Drosophila simulans to Drosophila serrata, a novel host, and compared parameter estimates with those from three naturally occurring Drosophila-Wolbachia associations believed to be of different ages. Transfected D. serrata showed strong incompatibility, low transmission efficiency, and an associated fitness deficit, and they would probably be unable to spread in nature. The comparisons generally supported the predicted evolution of a host-Wolbachia association. The parameters peculiar to any given host-Wolbachia association may determine whether the microbial strain can spread in that host.     

Incidence of Male-Killing Rickettsia spp. (α-Proteobacteria) in the Ten-Spot Ladybird Beetle Adalia decempunctata L. (Coleoptera: Coccinellidae)

The diversity of endosymbiotic bacteria that kill male host offspring during embryogenesis and their frequencies in certain groups of host taxa suggest that the evolution of male killing and the subsequent spread of male-killing symbionts are primarily determined by host life history characteristics. We studied the 10-spot ladybird beetle, Adalia decempunctata L. (Coleoptera: Coccinellidae), in which male killing has not been recorded previously, to test this hypothesis, and we also assessed the evolution of the male killer identified by DNA sequence analysis. Our results show that A. decempunctata harbors male-killing Rickettsia (α-proteobacteria). Male-killing bacteria belonging to the genus Rickettsia have previously been reported only for the congeneric two-spot ladybird beetle, Adalia bipunctata L. Phylogenetic analysis of Rickettsia DNA sequences isolated from different populations of the two host species revealed a single origin of male killing in the genus Rickettsia. The data also indicated possible horizontal transfer of symbionts between host species. In addition, A. bipunctata is known to bear at least four different male-killing symbionts in its geographic range two of which coexist in the two locations from which A. decempunctata specimens were obtained for the present study. Since only a single male-killing taxon was found in A. decempunctata, we assume that the two closely related ladybird beetle species must differ in the number and/or geographic distribution of male killers. We discuss the importance of these findings to our understanding of the evolution and dynamics of symbiotic associations between male-killing bacteria and their insect hosts.     

Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis

Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, α-DsbA1 and α-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein α-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of α-DsbA1 for both dithiol oxidants (E. coli DsbA, 12%) and disulfide isomerases (E. coli DsbC, 14%) is similar. We therefore sought to establish whether α-DsbA1 is an oxidant or an isomerase based on functional activity. The purified α-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that α-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified α-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins.