Vibrio cholerae Invasion Dynamics of the Chironomid Host Are Strongly Influenced by Aquatic Cell Density and Can Vary by Strain.

Cholera has been a human scourge since the early 1800s and remains a global public health challenge, caused by the toxigenic strains of the bacterium Vibrio cholerae. In its aquatic reservoirs, V. cholerae has been shown to live in association with various arthropod hosts, including the chironomids, a diverse insect family commonly found in wet and semiwet habitats. The association between V. cholerae and chironomids may shield the bacterium from environmental stressors and amplify its dissemination. However, the interaction dynamics between V. cholerae and chironomids remain largely unknown.  In this study, we developed freshwater microcosms with chironomid larvae to test the effects of cell density and strain on V. cholerae-chironomid interactions. Our results show that chironomid larvae can be exposed to V. cholerae up to a high inoculation dose (109 cells/mL) without observable detrimental effects. Meanwhile, interstrain variability in host invasion, including prevalence, bacterial load, and effects on host survival, was highly cell density-dependent. Microbiome analysis of the chironomid samples by 16S rRNA gene amplicon sequencing revealed a general effect of V. cholerae exposure on microbiome species evenness. Taken together, our results provide novel insights into V. cholerae invasion dynamics of the chironomid larvae with respect to various doses and strains. The findings suggest that aquatic cell density is a crucial driver of V. cholerae invasion success in chironomid larvae and pave the way for future work examining the effects of a broader dose range and environmental variables (e.g., temperature) on V. cholerae-chironomid interactions. IMPORTANCE Vibrio cholerae is the causative agent of cholera, a significant diarrheal disease affecting millions of people worldwide. Increasing evidence suggests that the environmental facets of the V. cholerae life cycle involve symbiotic associations with aquatic arthropods, which may facilitate its environmental persistence and dissemination. However, the dynamics of interactions between V. cholerae and aquatic arthropods remain unexplored. This study capitalized on using freshwater microcosms with chironomid larvae to investigate the effects of bacterial cell density and strain on V. cholerae-chironomid interactions. Our results suggest that aquatic cell density is the primary determinant of V. cholerae invasion success in chironomid larvae, while interstrain variability in invasion outcomes can be observed under specific cell density conditions. We also determined that V. cholerae exposure generally reduces species evenness of the chironomid-associated microbiome. Collectively, these findings provide novel insights into V. cholerae-arthropod interactions using a newly developed experimental host system.

Mining the diversity and functional profile of bacterial symbionts from the larvae of Chironomus circumdatus (bloodworms).

Chironomids are the most abundant aquatic insects in freshwater habitats that can survive in extreme conditions. In this study, as the microbiome provides extended genotype to the host to perform various functions, we explored the microbiota of the Chironomus circumdatus larvae to find out the putative role played by the symbiotic bacteria for the host. The metabarcoding analyses of the larvae revealed that the insect harbors 1771 phylotypes. Out of the various microbial communities found, the majority corresponded to the phyla Proteobacteria (52.59%) and Actinobacteria (20.56%), respectively. The midges also harbored Klebsiella (2.57%), Enterobacter (1.32%), Bacillus (2.29%), and Acinetobacter (2.13%) genera that are involved in detoxification of xenobiotics present in the water. The presence of radiation-resistant genera like Deinococcus, including bacterial species like radiodurans, a highly radiation-resistant bacterium, indicates its potential to support the host's ability to sustain in adverse environments. The functional profiling of the bacteria showed the relative abundance of many enzyme groups, such as transferases (40.62%), oxidoreductases (23.49%), and hydrolases (3.77%). The results indicate that the larvae harbor a considerable variety of bacteria that help the host adapt and survive in the polluted waters. The present study provides thorough insights into the microbiome of the C. circumdatus larvae that can be exploited for the bioremediation of certain pollutants through biomimetic strategies. It also gives us a wake-up call to take a good look at the guts of these disease-carrying insects' inabilities to spread deadly human diseases.

Identification of chironomid species as natural reservoirs of toxigenic Vibrio cholerae strains with pandemic potential.

Vibrio cholerae causes the fatal cholera diarrhea. Chironomids (Diptera; Chironomidae) are abundant in freshwater aquatic habitats and estuaries and are natural reservoirs of V. cholerae. Until now, only the non-O1/O139 serogroups of V. cholerae were identified in chironomids. Here, we explored whether chironomids are natural reservoirs of V. cholerae O1/O139 serogroups, which are associated with cholera endemics and pandemics. All four life stages of chironomids were sampled from two rivers, and a laboratory culture in Pune, India, and from a pond in Israel. In total, we analyzed 223 chironomid samples. The presence of V. cholerae O1/O139 serogroups was verified using molecular tools. Nine chironomid species were identified; of them, Chironomus circumdatus was the most abundant. The presence of V. cholerae serogroup O1 and the cholera toxin genes were detected in samples from all chironomid species. However, serogroup O139 was detected in only two chironomid species. Besides PCR to detect specific genes, a metagenomic analysis that was performed in three selected C. ramosus larvae, identified a list of virulence genes associated with V. cholerae. The findings provide evidence that chironomids are natural reservoirs of toxigenic V. cholerae O1/O139. Chironomid populations and V. cholerae show biannual peak patterns. A similar pattern is found for cholera epidemics in the Bengal Delta region. Thus, we hypothesize that monitoring chironomids in endemic areas of the disease may provide a novel tool for predicting and preventing cholera epidemics. Moreover, serogroup O139 was detected only in two chironomid species that have a restricted distribution in the Indian subcontinent, possibly explaining why the distribution of the O139 serogroup is limited.

First Gut Content Analysis of 4th Instar Midge Larvae (Diptera: Chronomidae) In Large-Scale Weirs Using a DNA Meta-Barcoding Approach.

Chironomidae larvae play an important role in the food chain of river ecosystems in Korea, where it is dominant. However, detailed information on the diet of Chironomidae larvae are still lacking. The purpose of this study was to identify the gut contents of 4th instar larvae of a Chironomidae inhabiting four large-scale weirs (Sejong Weir, Juksan Weir, Gangjeong-Goryeong Weir, and Dalseong Weir) using a DNA meta-barcoding approach. We found that dominant Operational Taxonomic Unit (OUT) was assigned to Paractinolaimus sp. (Nematoda), and the sub-dominant OTU was assigned to Dicrotendipes fumidus (Chironomidae). The most common OTUs among the individuals included phytoplankton, such as Tetrahymena sp., D. armatus, Pseudopediastrum sp., Tetradesmus dimorphus, Biddulphia tridens, and Desmodesmus spp. We calculated the selectivity index (E') and provided scientific evidence that Chironomidae larvae have a significant preference (E' > 0.5) for Desmodesmus armatus, E. minima, and T. dimorphus, while it does not show preference for other species found in its gut. Differences in physico-chemical factors, such as water quality, nutrients, Chl-a, and carbon concentrations, resulting from anthropogenic impacts (i.e., construction of large-scale weirs) as well as the particle size of prey organisms (small-sized single cell) and effects of chemicals (chemokinesis) could affect the feeding behavior of Chironomidae larvae.

Seasonal dynamics of Chironomus transvaalensis populations and the microbial community composition of their egg masses.

Chironomids (Diptera; Chironomidae) are the most abundant insects in freshwater environments and are considered natural reservoirs of Vibrio cholerae. We monitored the annual dynamics of chironomid populations along with their microbiota in order to better understand host-microbiota interactions. Chironomus transvaalensis populations peaked biannually in August and May-June. The composition of the endogenous bacterial communities of their egg masses clustered in two groups according to the sampling periods August-November and May-July. Nevertheless, a core bacterial community (43%) was present in all egg-mass samples. The most abundant phyla were: Proteobacteria, Firmicutes, Cyanobacteria and Bacteroidetes. The abundance of several genera (e.g. Rheinheimera and Pseudomonas) was positively correlated with C. transvaalensis population dynamics, while a predator-prey interaction was observed between the relative abundance of Vibrio OTUs and C. transvaalensis population size. Chironomids are known to tolerate toxic and stress conditions, and our results demonstrated that bacterial genera that may protect the insect under these conditions are present in the egg masses. After hatching, the first larval meal is the gelatinous matrix that surrounds the eggs. This meal contains a probiotic consortium that may protect the larva during its metamorphosis. The results provide important insights into the host-microbe interactions of chironomids.

A multi-year study following BACI design reveals no short-term impact of Bti on chironomids (Diptera) in a floodplain in Eastern Austria.

Short-term impacts of aerial application of Bacillus thuringiensis israelensis (Bti) on Culicidae and Chironomidae were investigated over several years in temporary waters of the Dyje and Morava floodplains in Eastern Austria. The sampling followed a Before-After-Control-Impact (BACI) approach with sampling dates immediately before and shortly after the application and was repeated for 3 years. To test for effects of the Bti treatment on the two Diptera families, linear mixed-effects models were used. Data analysis included the factors Before-After and Control-Impact as fixed effects, while general temporal and spatial variables were random effects. One hundred sixteen taxa of chironomids were identified. Abundance varied between 2 and 1125 larvae per m2, while culicid densities reached values of several 100 ind. per liter. Total culicid abundance significantly decreased after the Bti treatment, whereas no significant effects were found on the abundance of total chironomids and dominant chironomid subfamilies, tribes, and genera, on relative proportions of chironomid feeding guilds, diversity, and species composition. Further studies from this area are needed to extend the investigation over a period of several weeks in order to reveal possible delayed effects of the larvicide application.

Decreasing Bacillus thuringiensis israelensis sensitivity of Chironomus riparius larvae with age indicates potential environmental risk for mosquito control.

Mosquito control based on the use of Bacillus thuringiensis israelensis (Bti) is regarded as an environmental friendly method. However, Bti also affects non-target chironomid midges that are recognized as a central resource in wetland food webs. To evaluate the risk for different larval stages of Chironomus riparius we performed a test series of daily acute toxicity laboratory tests following OECD guideline 235 over the entire aquatic life cycle of 28 days. Our study is the first approach that performs an OECD approved test design with Bti and C. riparius as a standard organism in ecotoxicological testing. First-instar larvae of Chironomus riparius show an increased sensitivity towards Bti which is two orders of magnitude higher than for fourth instar larvae. Most EC50 values described in the literature are based on acute toxicity tests using third and fourth instar larvae. The risk for chironomids is underestimated when applying the criteria of the biocide regulation EU 528/2012 to our data and therefore the existing assessment approval is not protective. Possible impacts of Bti induced changes in chironomid abundances and community composition may additionally affect organisms at higher trophic levels, especially in spring when chironomid midges represent a key food source for reproducing vertebrates.

Histopathological effects of cypermethrin and Bacillus thuringiensis var. israelensis on midgut of Chironomus calligraphus larvae (Diptera: Chironomidae).

Pesticides are extensively used for the control of agricultural pests and disease vectors, but they also affect non-target organisms. Cypermethrin (CYP) is a synthetic pyrethroid used worldwide. Otherwise, bioinsecticides like Bacillus thuringiensis var. israelensis (Bti) have received great attention as an environmentally benign and desirable alternative. In order to evaluate the toxicity of those pesticides, Chironomus calligraphus was selected due to its high sensitivity to some toxicants. Third and fourth instars larvae were exposed to serial dilutions of CYP and Bti to determine LC50 values. In order to evaluate the potentially histopathological alterations as biomarkers, after 96-h of exposure, live larvae were fixed for histological analysis of the mid region of digestive tract. The 96-h LC50 values were 0.52 and 1.506µg/L for CYP and Bti, respectively. Midgut histological structure of the control group showed a single layer of cubical cells with microvilli in their apical surface and a big central nucleus. The midgut epithelium of larvae exposed to a low concentration of CYP (0.037µg/L) showed secretion activity and vacuolization while at high concentration (0.3µg/L) cells showed a greater disorganization and a more developed fat body. On the other hand, Bti caused progressive histological damage in this tissue. Chironomus calligraphus is sensitive to Bti and CYP toxicity like other Chironomus species. The histopathological alterations could be a valuable tool to assess toxicity mechanism of different pesticides.

A new sesquiterpene from the entomogenous fungus Phomopsis amygdali.

A new sesquiterpene, (+)-S-1-methyl-abscisic-6-acid (1), together with five known compounds, (+)-S-abscisic acid (2), fusicoccin J (3), 3α-hydroxyfusicoccin J (4), (R)-5-hydroxymethylmellein (5) and 4-hydroxyphenethyl acetate (6) was isolated from the fermentation extract of Phomopsis amygdali, an entomogenous fungus isolated from Call midge. Their structures were determined mainly by analysis of MS and NMR spectroscopic data. Compounds 1-6 were tested for antimicrobial activity against three plant pathogenic fungi: Gibberella zeae, Verticillium albo-atrum, and Fusarium nivale, and two bacteria: Escherichia coli and Pseudomonas aeruginosa 2033E. As a result, compounds 1-4 displayed antibacterial activity against Gram-negative P. aeruginosa 2033E, and the minimum inhibition concentration (MIC value) of 1-4 is 30 µg/mL, 58 µg/mL, 26 µg/mL, and 26 µg/mL, respectively.

Chironomid microbiome.

Chironomids are abundant insects in freshwater habitats. They undergo a complete metamorphosis of four life stages: eggs, larvae, and pupae in water, and a terrestrial adult stage. Chironomids are known to be pollution-tolerant, but little is known about their resistance mechanisms to toxic substances. Here we review current knowledge regarding the chironomid microbiome. Chironomids were found as natural reservoirs of Vibrio cholerae and Aeromonas spp. A stable bacterial community was found in the egg masses and the larvae when both culture-dependent and -independent methods were used. A large portion of the endogenous bacterial species was closely related to species known as toxicant degraders. Bioassays based on Koch's postulates demonstrated that the chironomid microbiome plays a role in protecting its host from toxic hexavalent chromium and lead. V. cholerae, a stable resident in chironomids, is present at low prevalence. It degrades the egg masses by secreting haemagglutinin/protease, prevents eggs from hatching, and exhibits host pathogen interactions with chironomids. However, the nutrients from the degraded egg masses may support the growth of the other microbiome members and consequently control V. cholerae numbers in the egg mass. V. cholerae, other chironomid endogenous bacteria, and their chironomid host exhibit complex mutualistic relationships.

Effects of benthic macrofauna bioturbation on the bacterial community composition in lake sediments.

Benthic macrofauna are considered to be an important part of the lacustrine ecosystem, and bioturbation may greatly affect the biogeochemical processes and microbial activities in sediments. In the present study, the bacterial community composition in sediments inhabited by 3 different types of benthic macrofauna (Corbicula fluminea, Chironomidae larvae, and tubificid worms) in the shallow and eutrophic Lake Taihu was studied to investigate the different effects of bioturbation on the composition of these communities. Microcosms were constructed, and culture-independent methods, including terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis, were performed to evaluate the bacterial communities. Analysis of similarities (ANOSIM) and multidimensional scaling (MDS) analysis of T-RFLP patterns demonstrated that differences in the bacterial community composition between the control and the macrofauna-inhabited sediments were not as great as expected, although the chemical properties of the sediments changed remarkably. Nevertheless, the dominant bacterial group in each type of macrofauna-inhabited sediment was different. Acidobacteria, Betaproteobacteria, and Deltaproteobacteria were the dominant bacterial groups in sediments inhabited by C. fluminea, tubificid worms, and Chironomidae larvae, respectively. The data obtained in this study are helpful for understanding the effects of bioturbation in a shallow, eutrophic lake.

Chironomidae bloodworms larvae as aquatic amphibian food.

Different species of chironomids larvae (Diptera: Chironomidae) so-called bloodworms are widely distributed in the sediments of all types of freshwater habitats and considered as an important food source for amphibians. In our study, three species of Chironomidae (Baeotendipes noctivagus, Benthalia dissidens, and Chironomus riparius) were identified in 23 samples of larvae from Belgium, Poland, Russia, and Ukraine provided by a distributor in Belgium. We evaluated the suitability of these samples as amphibian food based on four different aspects: the likelihood of amphibian pathogens spreading, risk of heavy metal accumulation in amphibians, nutritive value, and risk of spreading of zoonotic bacteria (Salmonella, Campylobacter, and ESBL producing Enterobacteriaceae). We found neither zoonotic bacteria nor the amphibian pathogens Ranavirus and Batrachochytrium dendrobatidis in these samples. Our data showed that among the five heavy metals tested (Hg, Cu, Cd, Pb, and Zn), the excess level of Pb in two samples and low content of Zn in four samples implicated potential risk of Pb accumulation and Zn inadequacy. Proximate nutritional analysis revealed that, chironomidae larvae are consistently high in protein but more variable in lipid content. Accordingly, variations in the lipid: protein ratio can affect the amount and pathway of energy supply to the amphibians. Our study indicated although environmentally-collected chironomids larvae may not be vectors of specific pathogens, they can be associated with nutritional imbalances and may also result in Pb bioaccumulation and Zn inadequacy in amphibians. Chironomidae larvae may thus not be recommended as single diet item for amphibians.

The protective role of endogenous bacterial communities in chironomid egg masses and larvae.

Insects of the family Chironomidae, also known as chironomids, are distributed worldwide in a variety of water habitats. These insects display a wide range of tolerance toward metals and organic pollutions. Bacterial species known for their ability to degrade toxicants were identified from chironomid egg masses, leading to the hypothesis that bacteria may contribute to the survival of chironomids in polluted environments. To gain a better understanding of the bacterial communities that inhabit chironomids, the endogenous bacteria of egg masses and larvae were studied by 454-pyrosequencing. The microbial community of the egg masses was distinct from that of the larval stage, most likely due to the presence of one dominant bacterial Firmicutes taxon, which consisted of 28% of the total sequence reads from the larvae. This taxon may be an insect symbiont. The bacterial communities of both the egg masses and the larvae were found to include operational taxonomic units, which were closely related to species known as toxicant degraders. Furthermore, various bacterial species with the ability to detoxify metals were isolated from egg masses and larvae. Koch-like postulates were applied to demonstrate that chironomid endogenous bacterial species protect the insect from toxic heavy metals. We conclude that chironomids, which are considered pollution tolerant, are inhabited by stable endogenous bacterial communities that have a role in protecting their hosts from toxicants. This phenomenon, in which bacteria enable the continued existence of their host in hostile environments, may not be restricted only to chironomids.

Lignocellulolytic enzymes and bacteria associated with the digestive tracts of Stenochironomus (Diptera: Chironomidae) larvae.

We analyzed the digestive activity of the enzymes that digest cellulose and hemicellulose and the bacterial community that is capable of hydrolyzing wood compounds in the digestive tracts of Stenochironomus (Diptera: Chironomidae) larvae, which are miners of decomposing submerged tree and bush branches. Based on quantification of reducing sugars, these larvae have a limited capacity for cellulose degradation but a good capacity for xylan hydrolysis. We isolated 31 types of colonies from two larval morphotypes, of which 19 tested positive for the capacity to hydrolyze at least one of the four substrates that were used as the main carbon source in the culture media. Their woody compound degradation capacity was assessed using colorimetric tests. The bacteria were identified by the analysis of the 16S rRNA gene. None of the bacteria were capable of degrading lignin. The genus Pseudomonas had the greatest species richness; Bacillus spp exhibited the greatest capacity for degrading the different substrates, and Sphingobium was found in both morphotypes. Microorganisms participate in the degradation of wood consumed by Stenochironomus larvae. This is the first report of lignocellulolytic bacteria and enzymes in the digestive tracts of mining chironomids.

New and rare Harpellales from Portugal and northwestern Iberian Peninsula: discovering the hidden mycobiota of Galicia-Tras-os-Montes region.

The species of Harpellales obtained from a survey of the gut fungi of aquatic arthropods in the Galaico-Portuguese region, also known as the Galicia-Trás-os-Montes Zone, are presented. New species include Orphella lusitanica, a close relative of O. helicospora in Plecoptera nymphs and Stachylina pleurospora, in Chironomiidae larvae, both from Galicia and Portugal. Tectimyces gallaecicus, in Ephemeroptera nymphs from Galicia, Spain, morphologically resembles the narrowly distributed T. robustus representing a genus known only from Europe. New geographical records from Portugal include Genistellospora homothallica, Graminella bulbosa, Harpella melusinae, Lancisporomyces vernalis, Legeriomyces ramosus, O. coronata, Simuliomyces microsporus and Smittium simulii. Also three rare taxa are reported from Galicia: Legeriomyces dolabrae and Baetimyces ancorae from Baetidae nymphs and Smittium inexpectans from Chironomidae larvae.

[Septicaemia of chironomid larvae (Diptera: Chironomidae) promoted by Bacillus cereus and B. thuringiensis].

Natural factors regulating the population of chironomids were studied. The bacteria Bacillus cereus were isolated from chironomids sampled from Kuyalnitskii Firth after epizooty of Chironomus sp., and bacteria Bacillus thuringiensis spp. israelensis (Bti) were isolated from dead larva of Chironomus plumosus sampled in the Sea of Azov (3-m depth). Bti were characterized by high insecticide activity on midges Anopheles messeae Fall., Aedes cireneus Mg., and Culex pipiens pipiens f. pipiens L.

Chironomid egg masses harbour the clinical species Aeromonas taiwanensis and Aeromonas sanarellii.

Bacteria of the genus Aeromonas are found worldwide in aquatic environments and may produce human infections. In 2010, two new clinical species, Aeromonas sanarellii and Aeromonas taiwanensis, were described on the basis of one strain recovered from wounds of hospitalized patients in Taiwan. So far, only four environmental isolates of A. sanarellii and one of A. taiwanensis have been recorded from waste water in Portugal and an additional clinical strain of A. taiwanensis from the faeces of a patient with diarrhoea in Israel. In the present study, strains belonging to these two species were identified from chironomid egg masses from the same area in Israel by sequencing the rpoD gene. This represents a new environmental habitat for these novel species. The first data on the virulence genes and antibiotic susceptibility are provided. The isolates of these two new species possess multiple virulence genes and are sensitive to amikacin, aztreonam, cefepime, cefoxatime, ceftazidime, ciprofloxacin, gentamicin, piperacillin-tazobactam, tigecycline, tobramycin, trimethoprim-sulfamethoxazole and imipenem. The key phenotypic tests for the differentiation of these new species from their closest relative Aeromonas caviae included the utilization of citrate, growth at 45 °C in sheep blood agar and acid production of cellobiose.

Bacterial community composition associated with chironomid egg masses.

Chironomids (Diptera: Chironomidae) are the most widely distributed and often the most abundant insect in freshwater. They undergo a complete metamorphosis of four life stages, of which the egg, larva, and pupae are aquatic and the adult is terrestrial. Chironomid egg masses were found to be natural reservoirs of Vibrio cholerae and Aeromonas species. To expand the knowledge of the endogenous bacterial community associated with chironomid egg masses, denaturing gradient gel electrophoresis and clone analysis of 16S rRNA gene libraries were used in this study. Bacterial community composition associated with chironomid egg masses was found to be stable among different sampling periods. Cloned libraries of egg masses revealed that about 40% of the clones were related to bacteria known to degrade various toxicants. These findings were further supported when bacterial species that showed resistance to different toxic metals were isolated from egg masses and larval samples. Chironomids are found under a wide range of water conditions and are able to survive pollutants. However, little is known about their protective mechanisms under these conditions. Chironomid egg masses are inhabited by a stable endogenous bacterial community, which may potentially play a role in protecting chironomids from toxicants in polluted environments. Further study is needed to support this hypothesis.

Effects of repeated field applications of two formulations of Bacillus thuringiensis var. israelensis on non-target saltmarsh invertebrates in Atlantic coastal wetlands.

Bacillus thuringiensis var. israelensis (Bti) is commonly used for selective control of larval populations of mosquitoes in coastal wetlands. A two year-study was implemented to investigate whether repeated treatments with Bti applied either as a liquid (VectoBac® 12AS) or a water-dispersible granule (VectoBac® WG) formulation may affect the abundance and diversity of non-target aquatic invertebrates in saltmarsh pools. Taxonomic composition of the invertebrate communities was typical of brackishwater intermittent ecosystems, with a dominance of annelids, crustaceans and nematocerans. Conditions were contrasted between the two years of the survey, both in terms of annual cumulative rainfall and rainfall distribution throughout the year. As a consequence, the hydroperiod and some other environmental characteristics associated with pool drying played a major role in the dynamics of the invertebrate community. In summer 2006, pool drying reduced the abundance of the polychaete worm Nereis diversicolor, of the amphipod crustacean Corophium volutator and of chironomid larvae. These taxa were able to recolonize rapidly the pools after flooding in September 2006. In 2007, rainfall was more regularly distributed across the year, and the pools did not get dry. Hydrozoans, Chironomini and Orthocladiinae larvae, and oligochaetes were more abundant in treated than in control pools, especially in VectoBac® WG-treated pools. No adverse effects of the treatments were shown on the abundance of N. diversicolor, C. volutator and midge larvae, suggesting that the availability of these food sources for birds was not negatively affected by Bti applications. It is concluded that, as currently performed in Western France coastal wetlands, land-based treatments of saltmarsh pools for larval mosquito control with Bti, used either as VectoBac® 12AS or VectoBac® WG, did not adversely impact non-target aquatic invertebrate communities.

Klastostachys, a new genus of Harpellales in Chironomidae larvae.

Klastostachys reflexa, a new genus and species combination in the Harpellales, is established herein based on Stachylina reflexa, which was described in 1988. This gut fungus was found attached to the peritrophic matrix of small bloodworms, Cryptochironomus sp. (Chironomidae), in the Rocky Mountains of Colorado, USA. Klastostachys resembles Stachylina, a genus common in Chironomidae, but the unbranched thalli of Klastostachys disarticulate and disperse with the trichospores remaining attached to their generative cells. This manner of dissemination is unusual among Harpellales, being noted also for Carouxella spp., but members of that genus have zygospores attached at one pole to the zygosporophore (Type IV), whereas Klastostachys zygospores are medially attached to the zygosporophore at right angles (Type I).