Dryocoetiops krivetsae sp. n. (Coleoptera: Curculionidae: Scolytinae: Dryocoetini), the northernmost species of the genus: conflicts between molecular and morphological data in the tribe Dryocoetini.

A new species of bark beetle, Dryocoetiops krivetsae Kerchev, Mandelshtam, Bykov et Ilinsky, sp. n., from the southern part of Primorsky Krai (Russian Far East), is described. This is the northernmost discovery of the Oriental genus Dryocoetiops Schedl, 1957, and the first record of the genus in the Russian fauna. Phylogenetic analysis of nuclear and mitochondrial genes i) confirms an independent lineage of Dryocoetiops krivetsae and ii) indicates numerous conflicts of genera relationships in the tribe Dryocoetini.

Genetic Evidence of an Isolation Barrier between Flea Subspecies of Citellophilus tesquorum (Wagner, 1898) (Siphonaptera: Ceratophyllidae).

This study investigated the relationship between two subspecies of the Citellophilus tesquorum flea, C. t. altaicus and C. t. sungaris, which are vectors of the bacterium Yersinia pestis that causes human plague across the vast territories of the Palearctic. Adult fleas were collected from 16 localities and 11 populations in 2019 and 2020. Specimens were morphologically verified for subspecies status and analysed for mitochondrial cytochrome c oxidase subunit I (COI) DNA, nuclear ribosomal cluster internal transcribed spacer 1 (ITS1) and ITS2, and Wolbachia-infection status. Our results demonstrated a genetic difference between C. t. altaicus and C. t. sungaris. According to mitochondrial data, the genetic distance between clades of C. t. altaicus and C. t. sungaris was comparable with the species divergence of the genus Callopsylla, which is closely related to Citellophilus. All studied populations of C. t. altaicus were Wolbachia-infected, whereas all studied populations of C. t. sungaris were symbiont-free. Data for ITS1 and ITS2 had much lower phylogenetic signals than mitochondrial data; however, diagnostic substitutions for C. t. altaicus and C. t. sungaris delimitation were also revealed. Analysis of a hardly accessible report on cross experiments allowed us to conclude the partial postzygotic isolation between these subspecies. Taken together, the molecular-genetic, geographical, morphological, and reproductive isolation findings verified that C. t. altaicus and C. t. sungaris subspecies could be considered as different species.

Narrow Genetic Diversity of Wolbachia Symbionts in Acrididae Grasshopper Hosts (Insecta, Orthoptera).

Bacteria of the Wolbachia genus are maternally inherited symbionts of Nematoda and numerous Arthropoda hosts. There are approximately 20 lineages of Wolbachia, which are called supergroups, and they are designated alphabetically. Wolbachia strains of the supergroups A and B are predominant in arthropods, especially in insects, and supergroup F seems to rank third. Host taxa have been studied very unevenly for Wolbachia symbionts, and here, we turn to one of largely unexplored insect families: Acrididae. On the basis of five genes subject to multilocus sequence typing, we investigated the incidence and genetic diversity of Wolbachia in 41 species belonging three subfamilies (Gomphocerinae, Oedipodinae, and Podisminae) collected in Turkey, Kazakhstan, Tajikistan, Russia, and Japan, making 501 specimens in total. Our results revealed a high incidence and very narrow genetic diversity of Wolbachia. Although only the strains belonging to supergroups A and B are commonly present in present, the Acrididae hosts here proved to be infected with supergroups B and F without A-supergroup variants. The only trace of an A-supergroup lineage was noted in one case of an inter-supergroup recombinant haplotype, where the ftsZ gene came from supergroup A, and the others from supergroup B. Variation in the Wolbachia haplotypes in Acrididae hosts within supergroups B and F was extremely low. A comprehensive genetic analysis of Wolbachia diversity confirmed specific features of the Wolbachia allelic set in Acrididae hosts. This result can help to elucidate the crucial issue of Wolbachia biology: the route(s) and mechanism(s) of Wolbachia horizontal transmission.

Unique Wolbachia strain wMelPlus increases heat stress resistance in Drosophila melanogaster.

Maternally inherited endosymbiotic bacterium Wolbachia infects Drosophila melanogaster populations worldwide. Its genetic diversity includes several closely related genotypes, which can be attributed to two main genotype groups: wMel and wMelCS. Here, we studied eight D. melanogaster lines carrying the nuclear background of wild type interbred Bi90 line and cytoplasmic backgrounds with or without Wolbachia of different origin, each of which belongs to wMelCS genotype group. We analyzed the effect these seven Wolbachia strains had on the heat stress resistance and dopamine metabolism in D. melanogaster females. Survival under heat stress (38°C, 3 h 30 min) was increased only in the line infected with bacteria of the wMelPlus strain. At the same time, the activity of alkaline phosphatase (an enzyme regulating the pool of dopamine precursor tyrosine) was increased under normal conditions in females infected with all strains under study and retained the response to heat stress typical for the uninfected line. Thus, we found the unique Wolbachia strain that provides an increase of the host stress resistance, and demonstrated that the mechanism of this resistance is not dopamine-mediated.

Appearances are deceptive: Three RNA viruses co-infected with the nucleopolyhedrovirus in host Lymantria dispar.

The virus infection, which visually looks like typical monoinfection, in fact may hide a great complex of different species. Without detailed analysis, we may miss the important interaction between pathogens, including new species. In the current study, we found the new species inside the mix of cubic and polyhedral occlusion bodies (OBs) isolated from the gypsy moth, Lymantria dispar L. (Ld). Transmission electron microscopy (TEM) revealed that into the one cadaver were OBs which belonged to baculovirus and cypoviruses. The baculovirus produced polyhedral OBs, while cypoviruses produced polyhedral and cubic OBs. Genomic analysis detected the multiple Ld nucleopolyhedroviruses, and cypoviruses were Hubei lepidoptera virus 3 and Dendrolimus punctatus cypovirus 1. This represents the first isolation of the Hubei lepidoptera virus 3 from the gypsy moth, proposed as "Lymantria dispar cypovirus 3". The RNAseq analysis also revealed the presence of Lymantria dispar iflavirus 1. The insecticidal activity of the mixed infection was comparable to that of typical baculovirus monoinfection. Thus, we demonstrate that i) the shape of OBs identified by light microscopy cannot be a robust indicator of viral species infecting the host; ii) only specific analysis may reveal the true composition of viral infection.

Sex-Specific Wolbachia Infection Patterns in Populations of Polygraphus proximus Blandford (Coleoptera; Curculionidae: Scolytinae).

Wolbachia symbionts are maternally inherited bacteria that are widely distributed among Arthropoda hosts. Wolbachia influence their host biology in diverse ways. They may induce reproductive abnormalities, protect hosts against pathogens and parasites, or benefit hosts through metabolic provisioning. The progeny of an infected female are ordinarily infected with Wolbachia; however, Wolbachia have no future in male host progeny because they cannot transmit the symbiont to the next generation. Here, we analyze native and invasive populations of the four-eyed fir bark beetle (Polygraphus proximus) for Wolbachia prevalence and symbiont genetic diversity. This species is a dangerous pest of Siberian fir (Abies sibirica) forests. The native range of P. proximus includes the territories of the Russian Far East, Japan, Korea, and Northeast China, whereas its invasive range includes West Siberia, with further expansion westward. Surprisingly, we revealed a difference in the patterns of Wolbachia prevalence for males and females. Infection rate and Wolbachia titers were higher in females than in males. ST-533, the only haplotype of Wolbachia supergroup B, was associated with a minimum of three out of the five described mitochondrial haplotypes.

Genetic evidence of broad spreading of Lymantria dispar in the West Siberian Plain.

Gypsy moth Lymantria dispar L. 1758 (Lepidoptera: Erebidae) is one of the most dangerous forest pests of the Holarctic region. Outbreaks of gypsy moth populations lead to significant defoliation of local forests. Within the vast territory of the West Siberian Plain, we noted the outbreak front movement in the north-east direction with a speed 100-200 km per year. The reason for the outbreak's movement is still unclear because L. dispar females are characterised by limited flight ability, which is not enough to support that movement per se. Herein, we analysed the mtDNA divergence pattern among L. dispar populations collected from the vast territory of the West Siberian Plain to determine the boundaries of populations and reveal the effect of the outbreak's front movement on mtDNA patterns of populations. The 590-bp region of the cytochrome oxidase subunit I gene of the mitochondrial genome was sequenced for 220 specimens that were collected from 18 localities along a transect line (~ 1400 km). Our results clearly show that the gypsy moth populations of the vast Siberian territory are not subdivided. This result can be explained by extensive genetic exchange among local populations. Taking into account that the flight ability of L. dispar females is rather limited, we suggest that spreading occurs through ballooning of early instar larvae. This hypothesis was confirmed by the coincidence of the outbreaks' movement direction with that of the dominant winds, complemented by the observation of ballooned larvae far from a forest edge.

Prevalence and genetic diversity of Wolbachia endosymbiont and mtDNA in Palearctic populations of Drosophila melanogaster.

BACKGROUND: Maternally inherited Wolbachia symbionts infect D. melanogaster populations worldwide. Infection rates vary greatly. Genetic diversity of Wolbachia in D. melanogaster can be subdivided into several closely related genotypes coinherited with certain mtDNA lineages. mtDNA haplotypes have the following global distribution pattern: mtDNA clade I is mostly found in North America, II and IV in Africa, III in Europe and Africa, V in Eurasia, VI is global but very rare, and VIII is found in Asia. The wMel Wolbachia genotype is predominant in D. melanogaster populations. However, according to the hypothesis of global Wolbachia replacement, the wMelCS genotype was predominant before the XX century when it was replaced by the wMel genotype. Here we analyse over 1500 fly isolates from the Palearctic region to evaluate the prevalence, genetic diversity and distribution pattrern of the Wolbachia symbiont, occurrence of mtDNA variants, and finally to discuss the Wolbachia genotype global replacement hypothesis. RESULTS: All studied Palearctic populations of D. melanogaster were infected with Wolbachia at a rate of 33-100%. We did not observe any significant correlation between infection rate and longitude or latitude. Five previously reported Wolbachia genotypes were found in Palearctic populations with a predominance of the wMel variant. The mtDNA haplotypes of the I_II_III clade and V clade were prevalent in Palearctic populations. To test the recent Wolbachia genotype replacement hypothesis, we examined three genomic regions of CS-like genotypes. Low genetic diversity was observed, only two haplotypes of the CS genotypes with a 'CCG' variant predominance were found. CONCLUSION: The results of our survey of Wolbachia infection prevalence and genotype diversity in Palearctic D. melanogaster populations confirm previous studies. Wolbachia is ubiquitous in the Palearctic region. The wMel genotype is dominant with local occurrence of rare genotypes. Together with variants of the V mtDNA clade, the variants of the 'III+' clade are dominant in both infected and uninfected flies of Palearctic populations. Based on our data on Wolbachia and mtDNA in different years in some Palearctic localities, we can conclude that flies that survive the winter make the predominant symbiont contribution to the subsequent generation. A comprehensive overview of mtDNA and Wolbachia infection of D. melanogaster populations worldwide does not support the recent global Wolbachia genotype replacement hypothesis. However, we cannot exclude wMelCS genotype rate fluctuations in the past.

Wolbachia symbionts in mosquitoes: Intra- and intersupergroup recombinations, horizontal transmission and evolution.

Many mosquitoes harbour Wolbachia symbionts that could affect the biology of their host in different ways. Evolutionary relationships of mosquitoes' Wolbachia infection, geographical distribution and symbiont prevalence in many mosquito species are not yet clear. Here, we present the results of Wolbachia screening of 17 mosquito species of four genera-Aedes, Anopheles, Coquillettidia and Culex collected from five regions of Eastern Europe and the Caucasus in 2012-2016. Based on multilocus sequence typing (MLST) data previously published and generated in this study, we try to reveal genetic links between mosquitoes' and other hosts' Wolbachia. The Wolbachia symbionts are found in Culex pipiens, Aedes albopictus and Coquillettidia richiardii and for the first time in Aedes cinereus and Aedes cantans, which are important vectors of human pathogens. Phylogenetic analysis demonstrated multiple origins of infection in mosquitoes although the one-allele-criterion approach revealed links among B-supergroup mosquito Wolbachia with allele content of lepidopteran hosts. The MLST gene content of strain wAlbA from the A-supergroup is linked with different ant species. Several cases of intersupergroup recombinations were found. One of them occurred in the wAlbaB strain of Aedes albopictus, which contains the coxA allele of the A-supergroup, whereas other loci, including wsp, belong to supergroup B. Other cases are revealed for non-mosquito symbionts and they exemplified genetic exchanges of A, B and F supergroups. We conclude that modern Wolbachia diversity in mosquitoes and in many other insect taxa is a recent product of strain recombination and symbiont transfers.

Molecular sexing of Lepidoptera.

We developed a universal method of Lepidoptera molecular sexing. The method is based on comparing the number of copies of the same gene in different sexes. Males of the majority of lepidopteran species have two Z chromosomes, whereas females have only one Z chromosome. Correspondingly, the number of copies of each gene located on this chromosome differs by two times between males and females. For quantitative estimation, we used qPCR. Via multiple alignment of the kettin (a Z chromosome gene) nucleotide sequences, we detected the most conserved fragment and designed primers with broad interspecies specificity for Lepidoptera. Using these primers, we successfully determined the sex of three lepidopteran species belonging to different superfamilies. The developed method is a simple, cost-effective and high-throughput technique for routine sexing. The sex of lepidopteran individuals can be examined at any developmental stage.

Drosophila female fertility and juvenile hormone metabolism depends on the type of Wolbachia infection.

Maternally inherited intracellular bacteria Wolbachia cause both parasitic and mutualistic effects on their numerous insect hosts, including manipulating the host reproductive system in order to increase the bacteria spreading in a host population, and increasing the host fitness. Here, we demonstrate that the type of Wolbachia infection determines the effect on Drosophila melanogaster egg production as a proxy for fecundity, and metabolism of juvenile hormone (JH), which acts as gonadotropin in adult insects. For this study, we used six D. melanogaster lineages carrying the nuclear background of interbred Bi90 lineage and cytoplasmic backgrounds with or without Wolbachia of different genotype variants. The wMelCS genotype of Wolbachia decreases egg production in infected D. melanogaster females in the beginning of oviposition and increases it later (from the sixth day after eclosion), whereas the wMelPop Wolbachia strain causes the opposite effect, and the wMel, wMel2 and wMel4 genotypes of Wolbachia do not show any effect on these traits compared with uninfected Bi90 D. melanogaster females. The intensity of JH catabolism negatively correlates with the fecundity level in the flies carrying both wMelCS and wMelPop Wolbachia The JH catabolism in females infected with genotypes of the wMel group does not differ from that in uninfected females. The effects of wMelCS and wMelPop infection on egg production can be levelled by the modulation of JH titre (via precocene/JH treatment of the flies). Thus, at least one of the mechanisms promoting the effect of Wolbachia on D. melanogaster female fecundity is mediated by JH.

Molecular diversity of Wolbachia in Lepidoptera: Prevalent allelic content and high recombination of MLST genes.

Wolbachia are common endosymbiotic bacteria of Arthropoda and Nematoda that are ordinarily transmitted vertically in host lineages through the egg cytoplasm. Despite the great interest in the Wolbachia symbiont, many issues of its biology remain unclear, including its evolutionary history, routes of transfer among species, and the molecular mechanisms underlying the symbiont's effect on its host. In this report, we present data relating to Wolbachia infection in 120 species of 13 Lepidoptera families, mostly butterflies, from West Siberian localities based on Multilocus sequence typing (MLST) and the wsp locus and perform a comprehensive survey of the distribution of Wolbachia and its genetic diversity in Lepidoptera worldwide. We observed a high infection incidence in the studied region; this finding is probably also true for other temperate latitude regions because many studied species have broad Palearctic and even Holarctic distribution. Although 40 new MLST alleles and 31 new STs were described, there was no noticeable difference in the MLST allele content in butterflies and probably also in moths worldwide. A genetic analysis of Wolbachia strains revealed the MLST allele core in lepidopteran hosts worldwide, viz. the ST-41 allele content. The key finding of our study was the detection of rampant recombination among MLST haplotypes. High rates of homologous recombination between Wolbachia strains indicate a substantial contribution of genetic exchanges to the generation of new STs. This finding should be considered when discussing issues related to the reconstruction of Wolbachia evolution, divergence time, and the routes of Wolbachia transmission across arthropod hosts.

Various Wolbachia genotypes differently influence host Drosophila dopamine metabolism and survival under heat stress conditions.

BACKGROUND: One of the most widespread prokaryotic symbionts of invertebrates is the intracellular bacteria of Wolbachia genus which can be found in about 50% of insect species. Wolbachia causes both parasitic and mutualistic effects on its host that include manipulating the host reproductive systems in order to increase their transmission through the female germline, and increasing the host fitness. One of the mechanisms, promoting adaptation in biological organisms, is a non-specific neuroendocrine stress reaction. In insects, this reaction includes catecholamines, dopamine, serotonin and octopamine, which act as neurotransmitters, neuromodulators and neurohormones. The level of dopamine metabolism correlates with heat stress resistance in Drosophila adults. RESULTS: To examine Wolbachia effect on Drosophila survival under heat stress and dopamine metabolism we used five strains carrying the nuclear background of interbred Bi90 strain and cytoplasmic backgrounds with different genotype variants of Wolbachia (produced by 20 backcrosses of Bi90 males with appropriate source of Wolbachia). Non-infected Bi90 strain (treated with tetracycline for 3 generations) was used as a control group. We demonstrated that two of five investigated Wolbachia variants promote changes in Drosophila heat stress resistance and activity of enzymes that produce and degrade dopamine, alkaline phosphatase and dopamine-dependent arylalkylamine N-acetyltransferase. What is especially interesting, wMelCS genotype of Wolbachia increases stress resistance and the intensity of dopamine metabolism, whereas wMelPop strain decreases them. wMel, wMel2 and wMel4 genotypes of Wolbachia do not show any effect on the survival under heat stress or dopamine metabolism. L-DOPA treatment, known to increase the dopamine content in Drosophila, levels the difference in survival under heat stress between all studied groups. CONCLUSIONS: The genotype of symbiont determines the effect that the symbiont has on the stress resistance of the host insect.

The impact of FOXO on dopamine and octopamine metabolism in Drosophila under normal and heat stress conditions.

The forkhead boxO transcription factor (FOXO) is a component of the insulin signalling pathway and plays a role in responding to adverse conditions, such as oxidative stress and starvation. In stressful conditions, FOXO moves from the cytosol to the nucleus where it activates gene expression programmes. Here, we show that FOXO in Drosophila melanogaster responds to heat stress as it does to other stressors. The catecholamine signalling pathway is another component of the stress response. In Drosophila, dopamine and octopamine levels rise steeply under heat, nutrition and mechanical stresses, which are followed by a decrease in the activity of synthesis enzymes. We demonstrate that the nearly twofold decline of FOXO expression in foxoBG01018 mutants results in dramatic changes in the metabolism of dopamine and octopamine and the overall response to stress. The absence of FOXO increases tyrosine decarboxylase activity, the first enzyme in octopamine synthesis, and decreases the enzymatic activity of enzymes in dopamine synthesis, alkaline phosphatase and tyrosine hydroxylase, in young Drosophila females. We identified the juvenile hormone as a mediator of FOXO regulation of catecholamine metabolism. Our findings suggest that FOXO is a possible trigger for endocrinological stress reactions.

Genetic integrity of four species of Leptidea (Pieridae, Lepidoptera) as sampled in sympatry in West Siberia.

In southern West Siberia, as many as four Leptidea Billberg, 1820 species are present sympatrically: Leptideaamurensis (Ménétriés, 1859), Leptideamorsei (Ménétriés, 1859), Leptideasinapis (Linnaeus, 1758) and Leptideajuvernica Williams, 1946. The two latter were recently recognised as nearly sibling species on morphological and molecular characters. Specimens intermediate as to their subtle diagnostic characters occurring in West Siberia and elsewhere were interpreted as resulted from limited introgression. This supposition was tested via populational morphological and molecular analysis of spring brood specimens of all the four species taken from a limited (4.5 × 0.2 km) area in the suburbs of Novosibirsk. The samples were analysed with respect to the genitalic morphology, external characters, three nuclear (CAD, H1 gene and ITS2) and one mitochondrial (COI) molecular markers, infection of the intracellular maternally inherited bacterial symbiont Wolbachia Hertig, 1836 and its wsp gene coding for a hypervariable surface protein. Interspecific variation of the nuclear CAD and ITS2 sequences and the mitochondrial COI gene in Leptideasinapis and Leptideajuvernica turned out concordant. The absence of molecular evidence of introgression suggests genetic integrity of these two species and allows their reliable identification by molecular characters. The genitalic (lengths of the saccus and valva) and external characters (wing pattern) of males overlap in Leptideasinapis and Leptideajuvernica, as identified by molecular markers and thus are not so helpful in actual species identification. Only the ductus bursae length showed no overlap and can be used for identification of females. The histone H1 gene appeared five times less variable over the four studied species than COI, and found to be identical in species Leptideasinapis and Leptideajuvernica. Wolbachia infection was found in all studied species. We identified three wsp variants of Wolbachia: 1) wsp-10 allele in Leptideaamurensis, Leptideasinapis, Leptideajuvernica; 2) a very similar wsp-687 allele in Leptideasinapis; and 3) wsp-688, highly divergent to the previous ones, in Leptideamorsei.

Coevolution of Drosophila melanogaster mtDNA and Wolbachia genotypes.

Maternally inherited microorganisms can influence the mtDNA pattern of variation in hosts. This influence is driven by selection among symbionts and can cause the frequency of mitochondrial variants in the population to eventually increase or decrease. Wolbachia infection is common and widespread in Drosophila melanogaster populations. We compared genetic variability of D. melanogaster mitotypes with Wolbachia genotypes among isofemale lines associated with different geographic locations and time intervals to study coevolution of the mtDNA and Wolbachia. Phylogenetic analysis of D. melanogaster mtDNA revealed two clades diverged in Africa, each associated with one of the two Wolbachia genotype groups. No evidence of horizontal transmission of Wolbachia between maternal lineages has been found. All the mtDNA variants that occur in infected isofemale lines are found in uninfected isofemale lines and vice versa, which is indicative of a recent loss of infection from some maternal fly lineages and confirms a significant role of Wolbachia in the D. melanogaster mtDNA pattern of variation. Finally, we present a comparative analysis of biogeographic distribution of D. melanogaster mitotypes all over the world.