Destabilization of the Bacterial Interactome Identifies Nutrient Restriction-Induced Dysbiosis in Insect Guts.

Stress-associated dysbiosis of microbiome can have several configurations that, under an energy landscape conceptual framework, can change from one configuration to another due to different alternating selective forces. It has been proposed-according to the Anna Karenina Principle-that in stressed individuals the microbiome are more dispersed (i.e., with a higher within-beta diversity), evidencing the grade of dispersion as indicator of microbiome dysbiosis. We hypothesize that although dysbiosis leads to different microbial communities in terms of beta diversity, these are not necessarily differently dispersed (within-beta diversity), but they form disrupted networks that make them less resilient to stress. To test our hypothesis, we select nutrient restriction (NR) stress that impairs host fitness but does not introduce overt microbiome selectors, such as toxic compounds and pathogens. We fed the polyphagous black soldier fly, Hermetia illucens, with two NR diets and a control full-nutrient (FN) diet. NR diets were dysbiotic because they strongly affected insect growth and development, inducing significant microscale changes in physiochemical conditions of the gut compartments. NR diets established new configurations of the gut microbiome compared to FN-fed guts but with similar dispersion. However, these new configurations driven by the deterministic changes induced by NR diets were reflected in rarefied, less structured, and less connected bacterial interactomes. These results suggested that while the dispersion cannot be considered a consistent indicator of the unhealthy state of dysbiotic microbiomes, the capacity of the community members to maintain network connections and stability can be an indicator of the microbial dysbiotic conditions and their incapacity to sustain the holobiont resilience and host homeostasis. IMPORTANCE Changes in diet play a role in reshaping the gut microbiome in animals, inducing dysbiotic configurations of the associated microbiome. Although studies have reported on the effects of specific nutrient contents on the diet, studies regarding the conditions altering the microbiome configurations and networking in response to diet changes are limited. Our results showed that nutrient poor diets determine dysbiotic states of the host with reduction of insect weight and size, and increase of the times for developmental stage. Moreover, the poor nutrient diets lead to changes in the compositional diversity and network interaction properties of the gut microbial communities. Our study adds a new component to the understanding of the ecological processes associated with dysbiosis, by disentangling consequences of diets on microbiome dysbiosis that is manifested with the disruption of microbiome networking properties rather than changes in microbiome dispersion and beta diversity.

Insights Into the Immune Response of the Black Soldier Fly Larvae to Bacteria.

In insects, a complex and effective immune system that can be rapidly activated by a plethora of stimuli has evolved. Although the main cellular and humoral mechanisms and their activation pathways are highly conserved across insects, the timing and the efficacy of triggered immune responses can differ among different species. In this scenario, an insect deserving particular attention is the black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae). Indeed, BSF larvae can be reared on a wide range of decaying organic substrates and, thanks to their high protein and lipid content, they represent a valuable source of macromolecules useful for different applications (e.g., production of feedstuff, bioplastics, and biodiesel), thus contributing to the development of circular economy supply chains for waste valorization. However, decaying substrates bring the larvae into contact with different potential pathogens that can challenge their health status and growth. Although these life strategies have presumably contributed to shape the evolution of a sophisticated and efficient immune system in this dipteran, knowledge about its functional features is still fragmentary. In the present study, we investigated the processes underpinning the immune response to bacteria in H. illucens larvae and characterized their reaction times. Our data demonstrate that the cellular and humoral responses in this insect show different kinetics: phagocytosis and encapsulation are rapidly triggered after the immune challenge, while the humoral components intervene later. Moreover, although both Gram-positive and Gram-negative bacteria are completely removed from the insect body within a few hours after injection, Gram-positive bacteria persist in the hemolymph longer than do Gram-negative bacteria. Finally, the activity of two key actors of the humoral response, i.e., lysozyme and phenoloxidase, show unusual dynamics as compared to other insects. This study represents the first detailed characterization of the immune response to bacteria of H. illucens larvae, expanding knowledge on the defense mechanisms of this insect among Diptera. This information is a prerequisite to manipulating the larval immune response by nutritional and environmental factors to increase resistance to pathogens and optimize health status during mass rearing.

Culturable bacteria in adults of a Southeast Asian black fly, Simulium tani (Diptera:Simuliidae).

Although the microbiome of blood-feeding insects serves an integral role in host physiology, both beneficial and pathogenic, little is known of the microbial community of black flies. An investigation, therefore, was undertaken to identify culturable bacteria from one of Malaysia's most common black flies, Simulium tani Takaoka and Davies, using 16S rDNA sequencing, and then evaluate the isolates for antibiotic resistance and virulence genes. A total of 20 isolates representing 11 bacterial species in four genera were found. Five isolates showed ß-hemolysis on Columbia agar, and virulence genes were found in three of these isolates. Some degree of resistance to six of the 12 tested antibiotics was found among the isolates. The baseline data from this study suggest rich opportunities for comparative studies exploring the diversity and roles of the microbiome of S. tani and other Southeast Asian black flies.

The general architecture of black fly-parasite interactions: Parasitism in lotic systems at a continental scale.

We examined the general architecture of interactions between stream-dwelling larval black flies (Diptera: Simuliidae) and their common parasites in 1736 collections across North America. Mermithid nematodes (family Mermithidae), microsporidia (phylum Microsporidia), and the fungus Coelomycidium simulii Debaisieux (phylum Blastocladiomycota) infected larval black flies. We found similar continental distributions for these three parasite taxa across North America. At least one of these taxa was represented in 42.2% of all black fly collections. Species interactions in ecological networks typically imply that each link between species is equally important. By employing quantitative measures of host susceptibilities and parasite dependencies, we provide a more complete structure for host-parasite networks. The distribution of parasite dependencies and host susceptibilities were right-skewed, with low values indicating that most dependencies (parasites) and susceptibilities (hosts) were weak. Although regression analysis between host frequency and parasite incidence were highly significant, frequency analysis suggested that the distributions of parasites differ significantly among the four most common and closely related (same subgenus) species of hosts. A highly significant pattern of nestedness in our bipartite host-parasite network indicated that specialized parasites (i.e., those that interact with few host species) tend to occur as subsets of the most common hosts.

Dual oxidase Duox and Toll-like receptor 3 TLR3 in the Toll pathway suppress zoonotic pathogens through regulating the intestinal bacterial community homeostasis in Hermetia illucens L.

Black soldier fly (BSF; Hermetia illucens L.) larvae can convert fresh pig manure into protein and fat-rich biomass, which can then be used as aquafeed for select species. Currently, BSF is the only approved insect for such purposes in Canada, USA, and the European Union. Pig manure could serve as a feed substrate for BSF; however, it is contaminated with zoonotic pathogens (e.g., Staphylococcus aureus and Salmonella spp.). Fortunately, BSF larvae inhibit many of these zoonotic pathogens; however, the mechanisms employed are unclear. We employed RNAi, qRT-PCR, and Illumina MiSeq 16S rDNA high-throughput sequencing to examine the interaction between two immune genes (Duox in Duox-reactive oxygen species [ROS] immune system and TLR3 in the Toll signaling pathway) and select pathogens common in pig manure to decipher the mechanisms resulting in pathogen suppression. Results indicate Bsf Duox-TLR3 RNAi increased bacterial load but decreased relative abundance of Providencia and Dysgonomonas, which are thought to be commensals in the BSF larval gut. Bsf Duox-TLR3 RNAi also inactivated the NF-κB signaling pathway, downregulated the expression of antimicrobial peptides, and diminished inhibitory effects on zoonotic pathogen. The resulting dysbiosis stimulated an immune response by activating BsfDuox and promoting ROS, which regulated the composition and structure of the gut bacterial community. Thus, BsfDuox and BsfTLR3 are important factors in regulating these key gut microbes, while inhibiting target zoonotic pathogens.

Isolation and characterization of a novel temperate bacteriophage from gut-associated Escherichia within black soldier fly larvae (Hermetia illucens L. [Diptera: Stratiomyidae]).

To gain insight into the presence and nature of prophages in the black soldier fly (BSF; Hermetia illucens L. [Diptera: Stratiomyidae]) gut, we isolated and characterized a novel, temperate Escherichia bacteriophage designated vB_EcoS_PHB10 (PHB10). Electron microscopy analysis revealed that phage PHB10 has a long, flexible, non-contractile tail and belongs to the family Siphoviridae. The phage was found to be stable over a wide range of temperatures (4-37 °C) and pH values (pH 5-9), and it lysed two out of 13 Escherichia strains tested. The genome of PHB10 contains genes encoding a putative transcriptional regulator and an integrase, and it shows a high degree of similarity to a region of the Enterobacter cloacae MBRL1077 genome. Induction experiments revealed that phage PHB10 could be induced by different gut substrates, suggesting that diet might be a potential regulator of lytic/lysogenic switches in commensal lysogens.

Molecular detection of Leucocytozoon (Apicomplexa: Haemosporida) in black flies (Diptera: Simuliidae) from Thailand.

Information regarding vector-parasite association is necessary for fully understanding the epidemiology of vector borne diseases yet, this information is lacking in the case of Leucocytozoonosis in the Oriental region, despite a high incidence of the disease. In this study, we used a molecular approach based on mitochondrial cytochrome b (cyt b) sequence to detect the parasite, Leucocytozoon, in potential black fly (Simuliidae) vectors in Thailand. A total of 404 wild caught black flies representing six morphological species of two subgenera were examined.- Gomphostilbia (Simulium asakoae complex, S. chumpornense) and Simulium (S. chamlongi, S. nodosum, S. nigrogilvum). Forty-four black fly specimens from two species of the Gomphostilbia were positive for Leucocytozoon. Most (35) of these were found in a village where high numbers of domestic chicken were kept, consistent with the possibility that chickens are a host of Leucocytozoon species found in black flies. Sixteen haplotypes were identified among 44 cyt b sequences. Comparisons of the sequences with previous reports revealed that the 11 haplotypes obtained in this study were identical or very similar to unknown Leucocytozoon found in infected domestic chickens. Four haplotypes are genetically similar to L. schoutedeni and one haplotype is genetically very different from existing cyt b sequences in public database. Our results indicate that two black fly species of the subgenus Gomphostilbia in Thailand are possible vectors of Leucocytozoon transmitted among poultry and wild birds in the country.

Vector species-specific association between natural Wolbachia infections and avian malaria in black fly populations.

Artificial infection of mosquitoes with the endosymbiont bacteria Wolbachia can interfere with malaria parasite development. Therefore, the release of Wolbachia-infected mosquitoes has been proposed as a malaria control strategy. However, Wolbachia effects on vector competence are only partly understood, as indicated by inconsistent effects on malaria infection reported under laboratory conditions. Studies of naturally-occurring Wolbachia infections in wild vector populations could be useful to identify the ecological and evolutionary conditions under which these endosymbionts can block malaria transmission. Here we demonstrate the occurrence of natural Wolbachia infections in three species of black fly (genus Simulium), which is a main vector of the avian malaria parasite Leucocytozoon. Prevalence of Leucocytozoon was high (25%), but the nature and magnitude of its association with Wolbachia differed between black fly species. Wolbachia infection was positively associated with avian malaria infection in S. cryophilum, negatively associated in S. aureum, and unrelated in S. vernum. These differences suggest that Wolbachia interacts with the parasite in a vector host species-specific manner. This provides a useful model system for further study of how Wolbachia influences vector competence. Such knowledge, including the possibility of undesirable positive association, is required to guide endosymbiont based control methods.

A survey of the mycobiota associated with larvae of the black soldier fly (Hermetia illucens) reared for feed production.

Feed security, feed quality and issues surrounding the safety of raw materials are always of interest to all livestock farmers, feed manufacturers and competent authorities. These concerns are even more important when alternative feed ingredients, new product developments and innovative feeding trends, like insect-meals, are considered. The black soldier fly (Hermetia illucens) is considered a good candidate to be used as feed ingredient for aquaculture and other farm animals, mainly as an alternative protein source. Data on transfer of contaminants from different substrates to the insects, as well as the possible occurrence of toxin-producing fungi in the gut of non-processed insects are very limited. Accordingly, we investigated the impact of the substrate/diet on the intestinal mycobiota of H. illucens larvae using culture-dependent approaches (microbiological analyses, molecular identification through the typing of isolates and the sequencing of the 26S rRNA D1/D2 domain) and amplicon-based next-generation sequencing (454 pyrosequencing). We fed five groups of H. illucens larvae at the third growing stage on two substrates: chicken feed and/or vegetable waste, provided at different timings. The obtained results indicated that Pichia was the most abundant genus associated with the larvae fed on vegetable waste, whereas Trichosporon, Rhodotorula and Geotrichum were the most abundant genera in the larvae fed on chicken feed only. Differences in the fungal communities were highlighted, suggesting that the type of substrate selects diverse yeast and mold genera, in particular vegetable waste is associated with a greater diversity of fungal species compared to chicken feed only. A further confirmation of the significant influence of diet on the mycobiota is the fact that no operational taxonomic unit common to all groups of larvae was detected. Finally, the killer phenotype of isolated yeasts was tested, showing the inhibitory activity of just one species against sensitive strains, out of the 11 tested species.

Description of microsporidia in simulids: molecular and morphological characterization of microsporidia in the larvae of Simulium pertinax Kollar (Diptera: Simuliidae).

INTRODUCTION: Microsporidia constitute the most common black fly pathogens, although the species' diversity, seasonal occurrence and transmission mechanisms remain poorly understood. Infections by this agent are often chronic and non-lethal, but they can cause reduced fecundity and decreased longevity. The objective of this study was to identify microsporidia infecting Simulium (Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba, State of São Paulo, Brazil, by molecular and morphological characterization. METHODS: Larvae were collected at a single point in a stream in a rural area of the city and were kept under artificial aeration until analysis. Polydispyrenia spp. infection was characterized by the presence of at least 32 mononuclear spores measuring 6.9 ± 1.0 × 5.0 ± 0.7 µm in persistent sporophorous vesicles. Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores measuring 4.5 × 3.5 µm in sporophorous vesicles. RESULTS: The molecular analysis confirmed the presence of microsporidian DNA in the 8 samples (prevalence of 0.51%). Six samples (Brazilian larvae) were related to Polydispyrenia simulii and Caudospora palustris reference sequences but in separate clusters. One sample was clustered with Amblyospora spp. Edhazardia aedis was the positive control taxon. CONCLUSIONS: Samples identified as Polydispyrenia spp. and Amblyospora spp. were grouped with P. simulii and Amblyospora spp., respectively, corroborating previous results. However, the 16S gene tree showed a considerable distance between the black fly-infecting Amblyospora spp. and the mosquito-infecting spp. This distance suggests that these two groups are not congeneric. Additional genomic region evaluation is necessary to obtain a coherent phylogeny for this group.

Description of microsporidia in simulids: molecular and morphological characterization of microsporidia in the larvae of Simulium pertinax Kollar (Diptera: Simuliidae)

Introduction Microsporidia constitute the most common black fly pathogens, although the species' diversity, seasonal occurrence and transmission mechanisms remain poorly understood. Infections by this agent are often chronic and non-lethal, but they can cause reduced fecundity and decreased longevity. The objective of this study was to identify microsporidia infecting Simulium (Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba, State of São Paulo, Brazil, by molecular and morphological characterization. Methods Larvae were collected at a single point in a stream in a rural area of the city and were kept under artificial aeration until analysis. Polydispyrenia spp. infection was characterized by the presence of at least 32 mononuclear spores measuring 6.9 ± 1.0 × 5.0 ± 0.7µm in persistent sporophorous vesicles. Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores measuring 4.5 × 3.5µm in sporophorous vesicles. Results The molecular analysis confirmed the presence of microsporidian DNA in the 8 samples (prevalence of 0.51%). Six samples (Brazilian larvae) were related to Polydispyrenia simulii and Caudospora palustris reference sequences but in separate clusters. One sample was clustered with Amblyospora spp. Edhazardia aedis was the positive control taxon. Conclusions Samples identified as Polydispyrenia spp. and Amblyospora spp. were grouped with P. simulii and Amblyospora spp., respectively, corroborating previous results. However, the 16S gene tree showed a considerable distance between the black fly-infecting Amblyospora spp. and the mosquito-infecting spp. This distance suggests that these two groups are not congeneric. Additional genomic region evaluation is necessary to obtain a coherent phylogeny for this group. .

Validation of the trichomycete Paramoebidium chattoni (Amoebidiales, Mesomycetozoea), a common and cosmopolitan black fly endosymbiont.

Paramoebidium, along with Amoebidium, constitute the Amoebidiales, which is phylogenetically embedded within the protist clade Ichthyosporea (Mesomycetozoea). However, the order is conventionally included within the trichomycetes, an ecological and polyphyletic group of arthropod-gut endosymbionts. Paramoebidium chattoni (Léger and Duboscq) Duboscq et al. is regarded as nomen nudum because the species was incorrectly published, and its taxonomic situation has never been addressed. Thus, P. chattoni is validated here with a description and neotypification to formalize the species and to guarantee the maintenance and correct use of its name. This common species has a wide geographical range and inhabits the digestive system of aquatic black fly larvae, where it frequently appears together with other gut endosymbionts, including P. curvum.

[Blackflies (Diptera: Simuliidae) of North of Armenia].

The fauna of blackflies of North Armenia is represented by 12 species from the genus Simulium. The species composition in the Tavush Region is most diverse (10 species) versus that in the Shirak (n = 5) and Lori (n = 3) Provinces, which is due to a wide variety of Simuliidae breeding sites. Among the bioregulators of blackflies, there are microsporidia of 4 species, cabbageworms (Mermithidae), and caddisflies of the genus Hydropsyche. There is a preponderance of microsporidia among the blackfly bioregulators. The highest percentage (60%) of the larvae infected with microsporidia is noted in the second half of summer, which is related to their environmental conditions.

Bacteria mediate oviposition by the black soldier fly, Hermetia illucens (L.), (Diptera: Stratiomyidae).

There can be substantial negative consequences for insects colonizing a resource in the presence of competitors. We hypothesized that bacteria, associated with an oviposition resource and the insect eggs deposited on that resource, serve as a mechanism regulating subsequent insect attraction, colonization, and potentially succession of insect species. We isolated and identified bacterial species associated with insects associated with vertebrate carrion and used these bacteria to measure their influence on the oviposition preference of adult black soldier flies which utilizes animal carcasses and is an important species in waste management and forensics. We also ascertained that utilizing a mixture of bacteria, rather than a single species, differentially influenced behavioral responses of the flies, as did bacterial concentration and the species of fly from which the bacteria originated. These studies provide insight into interkingdom interactions commonly occurring during decomposition, but not commonly studied.

[Biological agents for controlling the density of blood-sucking black flies (Diptera: Simuliidae) in the north of Armenia].

Biological agents were found to have high larvicidal activity against Simuliidae of two Bacillus thuringiensis spp. israelensis strains. To reduce the number of the pre-imago stages of black flies, the biological agent BLP-2477 should be used as most effective from an environmental point of view.

Gender-specific bacterial composition of black flies (Diptera: Simuliidae).

Although hematophagous black flies are well-known socioeconomic pests and vectors of disease agents, their associated bacteria are poorly known. A systematic analysis of the bacterial community associated with freshly emerged adult black flies of four North American species, using cultivation-independent molecular techniques, revealed 75 nonsingleton bacterial phylotypes. Although 17 cosmopolitan phylotypes were shared among host species, each fly species had a distinct bacterial profile. The bacterial composition, however, did not correlate strongly with the host phylogeny but differed between male and female flies of the same species from the same habitat, demonstrating that a group of insects have a gender-dependent bacterial community. In general, female flies harbor a less diverse bacterial community than do males. The anatomical locations of selected bacteria were revealed using fluorescence in situ hybridization. Understanding the physiological function of the associated bacterial community could provide clues for developing novel pest-management strategies.

The effect of seston on mortality of Simulium vittatum (Diptera: Simuliidae) from insecticidal proteins produced by Bacillus thuringiensis subsp. israelensis.

Water was collected from a site on the Susquehanna River in eastern Pennsylvania, where less-than-optimal black fly larval mortality had been occasionally observed after treatment with Bacillus thuringiensis subsp. israelensis de Barjac insecticidal crystalline proteins (Bti ICPs). A series of experiments was conducted with Simulium vittatum Zetterstedt larvae to determine the water related factors responsible for the impaired response to Bti ICPs (Vectobac 12S, strain AM 65-52). Seston in the water impaired the effectiveness of the ICPs, whereas the dissolved substances had no impact on larval mortality. Individual components of the seston then were exposed to the larvae followed by exposure to Bti ICPs. Exposure of larvae to selected minerals and nutritive organic material before ICP exposure resulted in no significant decrease in mortality. Exposure of larvae to silicon dioxide, cellulose, viable diatoms, and purified diatom frustules before Bti ICP exposure resulted in significant reductions in mortality. Exposure of larvae to purified diatom frustules from Cyclotella meneghiniana Kützing resulted in the most severe impairment of mortality after Bti ICP exposure. It is postulated that frustule-induced impairment of feeding behavior is responsible for the impairment of larval mortality.

Novel spirochetes isolated from mosquitoes and black flies in the Czech Republic.

During the years 1999-2002, a total of 4,898 individuals of 26 species of hematophagous insects (4,149 mosquitoes, 583 black flies, and 166 tabanid flies) was examined for the presence of spirochetes using dark-field microscopy. There was an overall recovery of spirochetes from the midguts of Culicidae and Simuliidae of 23.5% and 11.4%, respectively. Spirochetes were not detected in Tabanidae. Seven spirochetal strains have been successfully recovered from mosquitoes and black flies: BR149 (Culex pipiens), BR151 (Cx. pipiens), BR173 (Cx. pipiens), BR177 (Cx. pipiens), BR193 (Aedes cinereus), BR208 (Cx. pipiens), and BR231 (Simulium noelleri). The strains have been adapted to laboratory conditions (BSK-H Complete medium). Their preliminary determination based on 16S rRNA gene sequencing has shown that they differ from the Lyme disease spirochete Borrelia burgdorferi sensu lato as well as other members of the Order Spirochaetales indicating novel bacterial species in the Family Spirochaetaceae.

Phylogenetically distinct Wolbachia gene and pseudogene sequences obtained from the African onchocerciasis vector Simulium squamosum.

Wolbachia are intracellular bacteria mostly found in a diverse range of arthropods and filarial nematodes. They have been classified into seven distinct 'supergroups' and other lineages on the basis of molecular phylogenetics. The arthropod-infecting Wolbachia are usually regarded as reproductive parasites because they manipulate their host species' sexing system to enhance their own spread, and this has led to their investigation as potential agents of genetic control in medical entomology. We report 12 partial Wolbachia gene sequences from: aspC, aspS, dnaA, fbpA, ftsZ, GroEL, hcpA, IDA, rpoB, rpe, TopI and wsp as well as a single ftsZ pseudogene sequence, which have all been PCR-amplified from Simulium squamosum (Diptera: Simuliidae). To our knowledge this is the first such report from Simuliidae. Uninterrupted open-reading frame sequences were obtained from all 12 genes, covering approximately 6.2kb of unique DNA sequence. Phylogenetic analyses with the different coding genes gave consistent results suggesting that the Wolbachia sequences obtained here do not derive from any of the known Wolbachia supergroups or lineages. Consistent with a unique genetic status for the S. squamosumWolbachia, the hypervariable regions of the Wolbachia-specific wsp gene were distinct from all previous records in both sequence and length. As well as potential implications for newly emerging Wolbachia-based disease control methods, the results may be relevant to some problems experienced in the laboratory colonisation of Simulium damnosum sensu lato and why it is such a diverse species complex.

Morphological differences of symbiotic fungi Smittium culisetae (Harpellales: Legeriomycetaceae) in different Dipteran hosts.

Harpellales (Legeriomycetaceae, Zygomycota) or 'trichomycetes' are fungi that inhabit the digestive tracts of arthropods such as insects, millipedes, and crustaceans. In the current study we examined changes in 5 morphological characters of Smittium culisetae (Harpellales: Legeriomycetaceae) between the two dipteran (mosquito, black fly) hosts reared under 3 different temperatures (17, 22, 30 degrees C). Both host and temperature had a pervasive effect on the linear dimension of trichospores, their generative cells and hyphae width. At 30 degrees C the mean size of all 5 morphological characters were consistently larger in fungus taken from the mosquito host than from the black fly host. At 17 degrees C and 22 degrees C, however, there were no consistent patterns. The effect of host was so pronounced that it could be accurately determined which host S. culisetae colonised based on differences in linear morphology. Such changes in fungal morphology between hosts have important ramifications for the morphologically based taxonomy of this group.