Wolbachia dominance influences the Culex quinquefasciatus microbiota.

Microorganisms present in mosquitoes and their interactions are key factors affecting insect development. Among them, Wolbachia is closely associated with the host and affects several fitness parameters. In this study, the bacterial and fungal microbiota from two laboratory Culex quinquefasciatus isolines (wild type and tetracycline-cured) were characterized by metagenome amplicon sequencing of the ITS2 and 16S rRNA genes at different developmental stages and feeding conditions. We identified 572 bacterial and 61 fungal OTUs. Both isolines presented variable bacterial communities and different trends in the distribution of diversity among the groups. The lowest bacterial richness was detected in sugar-fed adults of the cured isoline, whereas fungal richness was highly reduced in blood-fed mosquitoes. Beta diversity analysis indicated that isolines are an important factor in the differentiation of mosquito bacterial communities. Considering composition, Penicillium was the dominant fungal genus, whereas Wolbachia dominance was inversely related to that of Enterobacteria (mainly Thorsellia and Serratia). This study provides a more complete overview of the mosquito microbiome, emphasizing specific highly abundant components that should be considered in microorganism manipulation approaches to control vector-borne diseases.

Human follicular mites: Ectoparasites becoming symbionts.

Most humans carry mites in the hair follicles of their skin for their entire lives. Follicular mites are the only metazoans tha continuously live on humans. We propose that Demodex folliculorum (Acari) represents a transitional stage from a host-injuring obligate parasite to an obligate symbiont. Here, we describe the profound impact of this transition on the genome and physiology of the mite. Genome sequencing revealed that the permanent host association of D. folliculorum led to an extensive genome reduction through relaxed selection and genetic drift, resulting in the smallest number of protein-coding genes yet identified among panarthropods. Confocal microscopy revealed that this gene loss coincided with an extreme reduction in the number of cells. Single uninucleate muscle cells are sufficient to operate each of the three segments that form each walking leg. While it has been assumed that the reduction of the cell number in parasites starts early in development, we identified a greater total number of cells in the last developmental stage (nymph) than in the terminal adult stage, suggesting that reduction starts at the adult or ultimate stage of development. This is the first evolutionary step in an arthropod species adopting a reductive, parasitic or endosymbiotic lifestyle. Somatic nuclei show underreplication at the diploid stage. Novel eye structures or photoreceptors as well as a unique human host melatonin-guided day/night rhythm are proposed for the first time. The loss of DNA repair genes coupled with extreme endogamy might have set this mite species on an evolutionary dead-end trajectory.

Mesostigmata diversity by manure type: a reference study and new datasets from southwestern Iran.

Manure-inhabiting Mesostigmata mites are important biological control agents of pest flies. However, the biodiversity of this mite community is mainly known from Europe and America, and especially from cattle manure. This study examined the diversity and abundance of Mesostigmata mites associated with various types of manure in an (intensive) agricultural region of the Middle East, i.e., the city Ahvaz and its suburbs, in southwest Iran. Mite samples were extracted from manure of cattle, buffalo, sheep, horse, poultry and quail in 30 livestock and poultry farms. In total, 40 species belonging to 24 genera and 16 families were identified. The most diverse families were Laelapidae with eight species, Macrochelidae with seven and Parasitidae with six. Macrocheles muscaedomesticae and Uroobovella marginata were the most widespread species, recorded in 28 and 27 out of 30 collection sites, respectively. Two species, M. sumbaensis and U. marginata, were found in all studied manures. Simpson's diversity index recorded the highest diversity in buffalo and sheep manure. Real and theoretical species richness (rarefaction curves) were congruent in number of individuals. The presence of seven species of Macrochelidae in the manure confirms that these are important predators of the house fly for the region of Ahvaz and its suburbs. Members of the Parasitidae were highly prevalent, with one species known as a specialized predator of house fly eggs. This work aims to encourage further studies on the diversity of Mesostigmata in these agricultural settings, and further continue assessing the feasibility of these mites as effective biocontrol agents of filth flies in different types of manure and from different corners of the world.

Ancient Human Genomes and Environmental DNA from the Cement Attaching 2,000-Year-Old Head Lice Nits.

Over the past few decades, there has been a growing demand for genome analysis of ancient human remains. Destructive sampling is increasingly difficult to obtain for ethical reasons, and standard methods of breaking the skull to access the petrous bone or sampling remaining teeth are often forbidden for curatorial reasons. However, most ancient humans carried head lice and their eggs abound in historical hair specimens. Here we show that host DNA is protected by the cement that glues head lice nits to the hair of ancient Argentinian mummies, 1,500-2,000 years old. The genetic affinities deciphered from genome-wide analyses of this DNA inform that this population migrated from north-west Amazonia to the Andes of central-west Argentina; a result confirmed using the mitochondria of the host lice. The cement preserves ancient environmental DNA of the skin, including the earliest recorded case of Merkel cell polyomavirus. We found that the percentage of human DNA obtained from nit cement equals human DNA obtained from the tooth, yield 2-fold compared with a petrous bone, and 4-fold to a bloodmeal of adult lice a millennium younger. In metric studies of sheaths, the length of the cement negatively correlates with the age of the specimens, whereas hair linear distance between nit and scalp informs about the environmental conditions at the time before death. Ectoparasitic lice sheaths can offer an alternative, nondestructive source of high-quality ancient DNA from a variety of host taxa where bones and teeth are not available and reveal complementary details of their history.

The impact of the decomposition process of shallow graves on soil mite abundance.

Burial of a cadaver results in a slower decomposition rate, due to more stable below-ground temperatures and restricted access to necrophagous insects. In such circumstances, analysis of the soil mesofauna, with emphasis on mites (Acari) may be more valuable in time-of-death estimations. The production of volatile organic compounds of cadaveric decay results in changes, especially in the soil pH, which in turn would affect the abundance and diversity of the associated mites. In general, the effects of decomposition and the consequently altered pH levels on the abundance of mites in shallow graves, as well as the effects of fluctuating above-ground environmental parameters (temperature, relative humidity, and precipitation) remain unknown. Here, we found that the decay of three pig cadavers buried in shallow graves (<30 cm below) caused a significant increase in the soil pH throughout decomposition, from neutral to alkaline. Cadaver decay attracted an abundance of mites: with 300 mites collected from the three pig cadavers compared to 129 from the control soil samples at the same depth. Mites rapidly became more abundant in cadaver-associated soils than in control soils after the fresh stage. Increasing soil pH had a positive impact on the abundance of mites in graves and there was a significant interaction between cadaver body temperature and soil pH. Above-ground fluctuations in temperature, relative humidity, and precipitation were found to have no significant direct effect on mite abundance in grave or control soils.

Assemblages of Acari in shallow burials: mites as markers of the burial environment, of the stage of decay and of body-cadaver regions.

The burial of a cadaver results in reduced arthropod activity and disruptions in colonisation patterns. Here, the distribution and diversity of mite taxa was studied across decomposition stages of shallowly buried pig carcasses (Sus scrofa domesticus). In total 300 mites (88 species) were collected from three pig shallow graves compared to 129 mites (46 species) from control (bare) soil samples at the same depth. A successional pattern of Acari higher taxa and families was observed, and species richness and biodiversity fluctuated throughout decomposition, whereas active decay showed the greatest biodiversity. Mesostigmata mites were the most abundant in 'cadaver soils' with a significant difference in the abundance of Parasitidae mites, whereas Oribatida mites (true soil mites) were the most abundant in control soils. Certain mite species were significantly associated with decay stages: Cornigamasus lunaris with 'bloated', Gamasodes spiniger with 'active', Eugamasus sp. and Lorryia reticulata with 'advanced', and Macrocheles matrius and Ramusella clavipectinata in 'dry'. Scheloribates laevigatus was a marker of bare soil at a shallow depth and Vulgoramasus remberti of buried decomposition, not specific to any decay stage. Analysis of mite assemblages associated with head, torso and posterior body showed that Parasitus evertsi and M. matrius are attracted to beneath the thighs, whereas L. reticulata to beneath the head. This study highlights the value of mites as indicator species of decomposition and its stages, confirming (1) a succession of Acari on buried remains and (2) species specificity to body regions.

Culex quinquefasciatus carrying Wolbachia is less susceptible to entomopathogenic bacteria.

In an attempt to evaluate the susceptibility of the mosquito Culex quinquefasciatus to bacterial agents, a population naturally infected with a Wolbachia pipientis wPipSJ native strain was tested against the action of three bacterial mosquitocides, Bacillus thuringiensis subsp. israelensis, Bacillus wiedmannii biovar thuringiensis and Lysinibacillus sphaericus. Tests were carried out on mosquito larvae with and without Wolbachia (controls). Cx. quinquefasciatus naturally infected with the native wPipSJ strain proved to be more resistant to the pathogenic action of the three mosquitocidal bacterial strains. Additionally, wPipSJ was fully characterised using metagenome-assembled genomics, PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) and MLST (MultiLocus Sequence Typing) analyses. This Wolbachia strain wPipSJ belongs to haplotype I, group wPip-III and supergroup B, clustering with other mosquito wPip strains, such as wPip PEL, wPip JHB, wPip Mol, and wAlbB; showing the southernmost distribution in America. The cytoplasmic incompatibility phenotype of this strain was revealed via crosses between wildtype (Wolbachia+) and antibiotic treated mosquito populations. The results of the tests with the bacterial agents suggest that Cx. quinquefasciatus naturally infected with wPipSJ is less susceptible to the pathogenic action of mosquitocidal bacterial strains when compared with the antibiotic-treated mosquito isoline, and is more susceptible to B. thuringiensis subsp. israelensis than to the other two mosquitocidal agents.

Co-haplotyping symbiont and host to unravel invasion pathways of the exotic pest Halyomorpha halys in Italy.

The brown marmorated stink bug Halyomorpha halys (Stål) is a globally invasive species that harbours the primary bacterial symbiont 'Candidatus Pantoea carbekii'. In this work, P. carbekii was used as another genetic marker to investigate the biodiversity and biogeographical patterns of this important pest, in native and newly invaded areas, especially in Italy. The correlation between the genetic structure of the symbiont and that of its host was studied through the analyses of one bacterial and one host marker, the putative pseudogene ΔybgF and the mitochondrial gene COI, respectively. As a result, five new P. carbekii haplotypes were identified, and an association pattern between host-symbiont haplotypes was observed. Host species showed higher haplotype diversity than symbiont, which can be expected in a long term host-symbiont association. Populations from the north-eastern Italy showed the highest values of genetic diversity for both markers, highlighting that this particular Italian area could be the result of multiple ongoing introductions. Moreover, some of the symbiont-host haplotypes observed were shared only by populations from north-eastern Italy and native areas, especially Japan, suggesting further introductions from this native country to Italy. Overall, our findings improve the understanding of the potential origin of multiple accidental introductions of H. halys in Italy.

Microscopic analysis of the microbiota of three commercial Phytoseiidae species (Acari: Mesostigmata).

Microbes associated with the external and internal anatomy of three commercially available predatory mite species-Phytoseiulus persimilis, Typhlodromips (= Amblyseius) swirskii, and Neoseiulus (= Amblyseius) cucumeris-were examined using light microscopy, confocal laser scanning microscopy and fluorescence in-situ hybridization (FISH). Four microbe morphotypes were observed on external body regions. These included three microfungi-like organisms (named T1, T2 and T3) and rod-shaped bacteria (T4). Morphotypes showed unique distributions on the external body regions and certain microbes were found only on one host species. Microfungi-like T1 were present in all three species whereas T2 and T3 were present in only P. persimilis and T. swirskii, respectively. T1 and T2 microbes were most abundant on the ventral structures of the idiosoma and legs, most frequently associated with coxae, coxal folds, ventrianal shields and epigynal shields. T3 microbes were most abundant on legs and dorsal idiosoma. T4 microbes were less abundant and were attached to epigynal shields of N. cucumeris and T. swirskii. Significant differences in distribution between batches suggest temporal fluctuations in the microbiota of phytoseiids in mass-reared systems. FISH showed bacteria within the alimentary tract, in Malpighian tubules and anal atria. These may aid absorption of excretory products or maintaining gut physiology. We suggest a mechanism by which microbes may be transmitted to offspring and throughout populations. This study aims to improve our knowledge of this poorly understood area and highlights the necessity of understanding the microbiota of Acari.

Mites (Acari) as a Relevant Tool in Trace Evidence and Postmortem Analyses of Buried Corpses.

This report interprets the presence of mite species in three clandestine graves in Europe, evaluating their potential use as trace evidence or markers. Grave 1 (Sweden): Two mite species Rhizoglyphus robini Claparède, 1869 and Parasitus loricatus (Wankel, 1861) were recovered from the surface of a body buried in a shallow grave in an area surrounded by trees, in close vicinity to house gardens. Grave 2 (Germany): Phoretic deutonymphs of Gamasodes spiniger (Trägårdh, 1910) were attached to an adult fly (Diptera: Sphaeroceridae) found within a shallow grave containing two human bodies covered in soil and dung. Grave 3 (France): P. loricatus were recovered from the soil around a body buried in a deep grave (80 cm under). In graves 1 and 3 both corpses were undergoing advanced decay and skeletization, the locations match with the subterranean habit of P. loricatus, highlighting the value of this species as a marker of graves or burials in soil and during late decomposition. R. robini is a soil mite that feeds on decayed roots and bulbs; this mite species confirms the location of the corpse within top soil, agreeing with a more specific type of superficial burial, a shallow grave. In case 2, the presence of both coprophiles, the mite G. spiniger and the carrier fly confirm association of remains with dung or animal feces. The three mite species are reported for the first time in human graves. There are no previous records of R. robini from Sweden.

First Report of the Ticks Haemaphysalis punctata Canestrini et Fanzago, 1878, Haemaphysalis parva (Neumann, 1897) and Dermacentor marginatus (Sulzer, 1776) (Acari, Amblyommidae) from Humans in Lebanon.

PURPOSE: Knowledge on ticks infesting humans is scarce for the middle East. In this work, tick specimens (Acari: Amblyommidae) infesting humans in Lebanon were identified. METHODS: Ticks that were found on humans were received in the Lebanese University, Faculty of Sciences. The specimens were preserved in alcohol for their further morphological identification. RESULTS: Three tick species were identified: a red sheep tick Haemaphysalis punctata Canestrini et Fanzago, 1878, a Mediterranean ear tick H. parva (Neumann, 1897), and an ornate sheep tick Dermacentor marginatus (Sulzer, 1776); all isolated from human hosts. CONCLUSION: This is the first report of Haemaphysalis punctata, H. parva and Dermacentor marginatus infesting humans from Central and North Lebanon.

Using human head lice to unravel neglect and cause of death.

Despite the common association of human lice with abandoned or neglected people, no procedure to assess pediculosis, aimed to detect signs of neglect, exists. Investigating the two most common forms of head louse infestation, regular and severe, we define lice-markers of neglect and develop a protocol and survey form to record and assess pediculosis. The study of head lice from a deceased victim of neglect helped unravel time-length since death, frequency of exposure to neglect and the cause and circumstances related to the death. Nit-clusters are markers of neglect, indicating length and frequency of neglect episodes. In the case study used here that culminated in the death of the victim, sustained abandonment started circa 2 years before discovery. The lice suggested that death was caused by overconsumption of a powerful calcium channel blocker, an antihypertensive, an excess of which in lice food supply (blood) stops oogenesis. Despite hosting thousands of adult females on the hair, lice reproduction stopped and nits were no longer developed or deposited on the hairs at the root end. This short distance of the shaft with no nits provided a time estimation of overdosing of almost 2 months before death.

Macrocheles species (Acari: Macrochelidae) associated with human corpses in Europe.

The biology of macrochelid mites might offer new venues for the interpretation of the environmental conditions surrounding human death and decomposition. Three human corpses, one from Sweden and two from Spain, have been analysed for the occurrence of Macrochelidae species. Macrocheles muscaedomesticae (Scopoli) females were associated with a corpse that was found in a popular beach area of southeast Spain. Their arrival coincides with the occurrence of one of their major carrier species, the filth fly Fannia scalaris, the activity of which peaks during mid-summer. Macrocheles glaber (Müller) specimens were collected from a corpse in a shallow grave in a forest in Sweden at the end of summer, concurrent with the arrival of beetles attracted by odours from the corpse. Macrocheles perglaber Filipponi and Pegazzano adults were sampled from a corpse found indoors in the rural surroundings of Granada city, south Spain. The phoretic behaviour of this species is similar to that of M. glaber, but it is more specific to Scarabaeidae and Geotrupidae dung beetles, most of which favour human faeces. Macrocheles muscaedomesticae is known from urban and rural areas and poultry farms, M. glaber from outdoors, particularly the countryside, whereas M. perglaber is known from outdoor, rural, and remote, potentially mountainous locations. Macrocheles muscaedomesticae and M. perglaber are reported for the first time from the Iberian Peninsula. This is the first record of M. perglaber from human remains.

Soil bulb mites as trace evidence for the location of buried money.

This study reports for the first time the use of soil micro-invertebrates, mites, as trace evidence to localise buried objects such as money. The case relates to a crime in Germany, where a large sum of banknotes had been hidden in an unknown location, likely abroad. In 2016, part of the money (approx. €500,000 in €500 value notes) was confiscated by the police. After analysis in the forensic lab, it was discovered that the notes were covered with small particles of a sort of debris, later identified as specimens of Rhizoglyphus howensis Manson, a non-European, nor Mediterranean species of root or bulb mites (Acaridae: Rhizoglyphinae). The restricted biogeographic distribution of R. howensis, originated in unspoiled forest soil in the Australasian region, limited the search for the money to the areas visited by the perpetrators during their trips to the region. Rhizoglyphus howensis biology provided further clues on the whereabouts of the banknotes, as they are specialist plant feeders, exclusively feeding on seeds of palm trees and on roots of Quercus patula in the Australasian region. This report aims to highlight the importance of the correct identification of the microscopic organisms associated with a crime scene and the immediate retrieval of micro-invertebrate trace evidence. This is the first record of R. howensis from Europe, and from banknotes.

Comparative analysis of bones, mites, soil chemistry, nematodes and soil micro-eukaryotes from a suspected homicide to estimate the post-mortem interval.

Criminal investigations of suspected murder cases require estimating the post-mortem interval (PMI, or time after death) which is challenging for long PMIs. Here we present the case of human remains found in a Swiss forest. We have used a multidisciplinary approach involving the analysis of bones and soil samples collected beneath the remains of the head, upper and lower body and "control" samples taken a few meters away. We analysed soil chemical characteristics, mites and nematodes (by microscopy) and micro-eukaryotes (by Illumina high throughput sequencing). The PMI estimate on hair 14C-data via bomb peak radiocarbon dating gave a time range of 1 to 3 years before the discovery of the remains. Cluster analyses for soil chemical constituents, nematodes, mites and micro-eukaryotes revealed two clusters 1) head and upper body and 2) lower body and controls. From mite evidence, we conclude that the body was probably brought to the site after death. However, chemical analyses, nematode community analyses and the analyses of micro-eukaryotes indicate that decomposition took place at least partly on site. This study illustrates the usefulness of combining several lines of evidence for the study of homicide cases to better calibrate PMI inference tools.

Author Correction: Comparison of bacterial microbiota of the predatory mite Neoseiulus cucumeris (Acari: Phytoseiidae) and its factitious prey Tyrophagus putrescentiae (Acari: Acaridae).

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Comparison of bacterial microbiota of the predatory mite Neoseiulus cucumeris (Acari: Phytoseiidae) and its factitious prey Tyrophagus putrescentiae (Acari: Acaridae).

Neoseiulus cucumeris is a predatory mite used for biological control of arthropod pests. Mass-reared predators are fed with factitious prey mites such as Tyrophagus putrescentiae. Although some information on certain endosymbionts of N. cucumeris and T. putrescentiae exists, it is unclear whether both species share bacterial communities. The bacterial communities in populations of predator and prey mites, as well as the occurence of potential acaropathogenic bacteria were analyzed. The comparisons were based on the following groups: (i) N. cucumeris mass-production; (ii) N. cucumeris laboratory population with disease symptoms; (iii) T. putrescentiae pure populations and; (iv) T. putrescentiae from rearing units of N. cucumeris. Only 15% of OTUs were present in all samples from predatory and prey mite populations (core OTUs): the intracellular symbionts Wolbachia, Cardinium, plus other Blattabacterium-like, Solitalea-like, and Bartonella-like symbionts. Environmental bacteria were more abundant in predatory mites, while symbiotic bacteria prevailed in prey mites. Relative numbers of certain bacterial taxa were significantly different between the microbiota of prey mites reared with and without N. cucumeris. No significant differences were found in the bacterial communities of healthy N. cucumeris compared to N. cucumeris showing disease symptoms. We did not identify any confirmed acaropathogenic bacteria among microbiota.

The ghost sex-life of the paedogenetic beetle Micromalthus debilis.

Genetic and sexual systems can be evolutionarily dynamic within and among clades. However, identifying the processes responsible for switches between, for instance, sexual and asexual reproduction, or cyclic and non-cyclic life histories remains challenging. When animals evolve parthenogenetic reproduction, information about the sexual mating system becomes lost. Here we report an extraordinary case where we have been able to resurrect sexual adults in a species of beetle that reproduces by parthenogenetic paedogenesis, without the production of adults. Via heat treatment, we were able to artificially induce adult beetles of Micromalthus debilis in order to describe its pre-paedogenetic mating system. Adults showed a highly female biased sex ratio, out-breeding behaviour, and sex-role reversal. Paedogenetic larvae of Micromalthus are infected with the endosymbiotic bacteria Rickettsia and Wolbachia. Clear signs of vestigialization in adults are concurrent with the loss of adults. Our data suggest an ancient female sex ratio bias that predates the loss of adults, perhaps associated with endosymbionts. We propose a model for the transition from a haplodiploid cyclical parthenogenetic life history to parthenogenetic paedogenesis. Paedogenetic development induces a new mechanism of sex ratio bias in midges, wasps and beetles.

Culex pipiens Development Is Greatly Influenced by Native Bacteria and Exogenous Yeast.

Culex pipiens is the most cosmopolitan mosquito of the Pipiens Assemblage. By studying the nature of interactions between this species and microorganisms common to its breeding environment we can unravel important pitfalls encountered during development. We tested the survival rate of larval stages, pupae and adults of a Cx. pipiens colony exposed to a variety of microorganisms in laboratory conditions and assessed the transmission to offspring (F1) by those organisms that secured development up to adulthood. Three complementary experiments were designed to: 1) explore the nutritional value of yeasts and other microorganisms during Cx. pipiens development; 2) elucidate the transstadial transmission of yeast to the host offspring; and 3) to examine the relevance of all these microorganisms in female choice for oviposition-substratum. The yeast Saccharomyces cerevisiae proved to be the most nutritional diet, but despite showing the highest survival rates, vertical transmission to F1 was never confirmed. In addition, during the oviposition trials, none of the gravid females was attracted to the yeast substratum. Notably, the two native bacterial strains, Klebsiella sp. and Aeromonas sp., were the preferred oviposition media, the same two bacteria that managed to feed neonates until molting into 2nd instar larvae. Our results not only suggest that Klebsiella sp. or Aeromonas sp. serve as attractants for oviposition habitat selection, but also nurture the most fragile instar, L1, to assure molting into a more resilient stage, L2, while yeast proves to be the most supportive diet for completing development. These experiments unearthed survival traits that might be considered in the future development of strategies of Cx. pipiens control. These studies can be extended to other members of the Pipiens Assemblage.