Culturable and VBNC <Emphasis Type="Italic">Vibrio cholerae</Emphasis>: Interactions with Chironomid Egg Masses and Their Bacterial Population

Vibrio cholerae, the etiologic agent of cholera, is autochthonous to various aquatic environments. Recently, it was found that chironomid (nonbiting midges) egg masses serve as a reservoir for the cholera bacterium and that flying chironomid adults are possible windborne carriers of V. cholerae non-O1 non-O139. Chironomids are the most widely distributed insect in freshwater. Females deposit egg masses at the water's edge, and each egg mass contains eggs embedded in a gelatinous matrix. Hemagglutinin/protease, an extracellular enzyme of V. cholerae, was found to degrade chironomid egg masses and to prevent them from hatching. In a yearly survey, chironomid populations and the V. cholerae in their egg masses followed phenological succession and interaction of host–pathogen population dynamics. In this report, it is shown via FISH technique that most of the V. cholerae inhabiting the egg mass are in the viable but nonculturable (VBNC) state. The diversity of culturable bacteria from chironomid egg masses collected from two freshwater habitats was determined. In addition to V. cholerae, representatives of the following genera were isolated: Acinetobacter, Aeromonas, Klebsiella, Shewanella, Pseudomonas, Paracoccus, Exiguobacterium, and unidentified bacteria. Three important human pathogens, Aeromonas veronii, A. caviae, and A. hydrophila, were isolated from chironomid egg masses, indicating that chironomid egg masses may be a natural reservoir for pathogenic Aeromonas species in addition to V. cholerae. All isolates of V. cholerae were capable of degrading chironomid egg masses. This may help explain their host–pathogen relationship with chironomids. In contrast, almost none of the other bacteria that were isolated from the egg masses possessed this ability. Studying the interaction between chironomid egg masses, the bacteria inhabiting them, and V. cholerae could contribute to our understanding of the nature of the V. cholerae–egg mass interactions.     

Novel insights into Haemagglutinin Protease (HAP) gene regulation in Vibrio cholerae

Quorum sensing is the phenomenon, whereby bacteria use signal molecules to communicate with each other. For example, to establish a successful infection, pathogenic bacteria become virulent only when they reach a certain local concentration in their host. Bassler and others have highlighted the surprising observation that quorum sensing seems to repress Vibrio cholerae virulence factor expression (e.g. cholera toxin), in contrast to what has been observed for virulence gene expression in other bacteria. Here, I present a novel insight that may clarify the way V. cholerae quorum‐sensing signals regulate its genes. Chironomids (Diptera; Chironomidae), which occur worldwide and are frequently the insect found most abundantly in fresh water bodies, are natural reservoirs of V. cholerae. Quorum‐sensing signals in V. cholerae up‐regulate the production of an extracellular enzyme, haemagglutinin protease (HAP), which degrades chironomid egg masses and prevents the eggs from hatching. HAP, therefore, is a virulence factor against chironomids. Indeed, in a survey carried out over the course of a year, V. cholerae and chironomids showed a pattern that mirrored the dynamics of predator‐prey populations. Globally, the numbers of chironomids are much larger than those of humans, so quorum‐sensing signals of V. cholerae and HAP gene regulation should be understood with regard to their role in chironomids rather than humans. Further research is needed to understand the role of cholera toxin in the environmental existence of V. cholerae.     

Chironomid Egg Masses as a Natural Reservoir of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Non-O1 and Non-O139 in Freshwater Habitats

Cholera is a diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, and an estimated 120,000 deaths from cholera occur globally every year. The natural reservoir of the bacterium is environmental. A recent report indicated an association between V. cholerae and chironomid egg masses. Chironomids, the non-biting midges (Diptera; Chironomidae), are the most widely distributed and frequently the most abundant insects in freshwater. Females attach egg masses, each containing hundreds of eggs encased in a layer of gelatin, to the waters edge where bacteria are abundant and may encounter the nutrient-rich substrate. Here we report the isolation of non-O1 and non-O139 V. cholerae from chironomid egg masses from different freshwater bodies in Israel, India, and Africa. In a yearly survey in Israel, chironomid populations were found to peak biannually, and it seemed that those peaks were followed by subsequent bacterial growth and disappearance during the winter in the Mediterranean region. The bacterial population rose as water temperature surpassed 25°C. Thirty-five different serogroups of V. cholerae were identified among the bacteria isolated from chironomids, demonstrating population heterogeneity. Two strains of V. cholerae O37 and O201 that were isolated from chironomid egg masses in Zanzibar Island were NAG-ST positive. Our findings support the hypothesis that the association found between chironomids and the cholera bacteria is not a rare coincidence, indicating that chironomid egg masses may serve as yet another potential reservoir for V. cholerae.     

Food‐availability dependent premature metamorphosis in the bean blister beetle Epicauta gorhami (Coleoptera: Meloidae), a hypermetamorphic insect that feeds on grasshopper eggs in the larval stage

Larvae of the bean blister beetle, Epicauta gorhami, feed on only grasshopper eggs and undergo hypermetamorphosis with pseudopupal diapause in the fifth instar. Whether E. gorhami larvae enter pseudopupal diapause or pupate directly from the fourth instar is controlled by temperature and photoperiod. In nature, larvae are confronted with a significant variation in the availability of food, suggesting the possibility that feeding conditions may also affect the diapause incidence. Here, we addressed this issue by changing the feeding conditions in the fourth instar under conditions of 16 h light : 8 h dark (LD 16 : 8) at 25°C. Food deprivation reduced the length of instar and increased the tendency to pupate, leading to the early eclosion of a small adult. Even non‐feeding fourth‐instar larvae pupated. Regardless of the timing of food deprivation, the post‐feeding larval period was constant and equivalent to that of ad libitum‐fed larvae, suggesting that premature exhaustion of the food supply triggers the initiation of pupation. In agreement with these results, when larvae were fed on intact grasshopper egg pods of various sizes from four species, those that fed on smaller egg pods had a decreased tendency to pseudopupate (i.e., to enter diapause). Food‐deprived larvae showed a clearer photoperiodic response and had a shorter critical day‐length. Thus, in E. gorhami, feeding conditions do not affect pupation success, but do affect the tendency to pupate or pseudopupate. This is the first report of the occurrence of premature pupation in carnivorous insects. We discuss our findings in the context of the natural history and behavioral ecology of E. gorhami.     

Phylloplane bacteria increase the negative impact of food limitation on insect fitness

1. When populations of herbivorous insects increase in density, they can alter the quantity or quality of their food. The impacts of diet‐related stressors on insect fitness have been investigated singly, but not simultaneously. 2. Foliage quantity and quality of red alder, Alnus rubra, were manipulated together with the presence of non‐entomopathogenic phylloplane bacteria to investigate their impacts, singly and in combination, on survival, pupal mass, growth rate, fecundity and egg quality of a cyclic forest insect, the western tent caterpillar, Malacosoma californicum pluviale. 3. Food limitation (half food) had strong negative impacts on all life‐history traits. When the larvae were fed continuously, however, neither ingesting phylloplane bacteria nor eating leaves from damaged branches (induced foliage) affected survival. In the half‐food treatment, ingesting bacteria further increased mortality, while feeding on induced foliage improved survival. 4. Growth rate and pupal mass of both sexes were reduced for larvae with food limitation compared with continuously fed insects and this was exacerbated when the larvae also ate bacteria‐treated leaves. A combination of bacteria and induced foliage also reduced larval growth rate by 5% in the full‐food treatment. 5. Fecundity (eggs per egg mass) was 2.7 times greater in full‐food than in food‐limited treatments but neither phylloplane bacteria nor plant induction had an effect. Insects fed induced foliage produced smaller eggs. Overall, there was no evidence of a three‐way interaction between the three stressors, although there were negative synergistic effects, primarily between food limitation and the ingestion of phylloplane bacteria.     

Stay or move: how Bt‐susceptible Helicoverpa armigera neonates behave on Bt cotton plants

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae occasionally have been reported to survive at management threshold levels in fields of Bollgard II^® cotton, Gossypium hirsutum L. (Malvaceae). The pattern and degree of larval survival is not easily predicted but depends on the ability of first instars to establish on host plants. Experiments were conducted with Bacillus thuringiensis Berliner (Bt)‐susceptible and Bt‐resistant larvae of H. armigera to understand how physiologically Bt‐susceptible H. armigera survive on Bt cotton plants, and examine how their first meal influences survival rates. In assays using cotton plant parts, both strains of larvae displayed similar tendencies to drop‐off specific plant parts of Bt and non‐Bt cotton. However, significantly more Bt‐susceptible larvae dropped off young leaves, mature leaves, and squares of Bt cotton compared to non‐Bt cotton plants. Egg cannibalism significantly improved the survival of Bt‐susceptible H. armigera larvae on Bt cotton plants. Larvae were more likely to eat live aged eggs, than newly laid or dead eggs. Survival significantly improved when larvae cannibalized eggs before feeding on Bt leaves. The behavior of Bt‐susceptible larvae with respect to drop‐off and egg cannibalism may help enhance their survival on Bt cotton plants.     

Food spectra of the farm juveniles of the Chum Salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> (Salmoniformes,Salmonidae) in the Ryazanovka River (Southern Primor’e)

Qualitative data are presented on the diet of juvenile chum salmon Oncorhynchus keta released from the Ryazanovka experimental fish hatchery farm situated in the south of Primor’e Territory. The stomach fullness, frequency of occurrence of food items, their average weight value, and selectivity are estimated. Food spectra of chum salmon juveniles during downstream migration comprised representatives of 51 taxa of bottom freshwater invertebrates and terrestrial insects. The bulk of the diet consisted of larvae of aquatic insects—chironomids, other Diptera, and mayflies. Their diversity increased with the growth of fry. Fry of chum salmon had a wide spectrum of feeding preference, favorite food items were larvae and pupae of chironomids.     

Potential for passive internal dispersal: eggs of an aquatic leaf beetle survive passage through the digestive system of mallards

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Life histories of closely related amphidromous and non‐migratory fish species: a trade‐off between egg size and fecundity

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The reproductive and feeding biology of Schilbe intermedius Rüppell in the Okavango Delta,Botswana

The reproductive and feeding biology of 314 Schilbe intermedius Rüppell 1832 collected in the Okavango Delta, Botswana, between April 1985 and February 1986 during low, intermediate and high water level were investigated. S. intermedius matures at 130 mm standard length and is a monocyclic spawner. Females predominate in the population and have an average fecundity of 27107 eggs. In the northern riverine floodplain of the Delta fish spawn in mid-summer during the peak flood. In the southern drainage rivers fish spawn in spring, which is two to three months after the arrival of the winter flood waters. S. intermedius is an opportunistic predator with a diet consisting largely of fish 41%, aquatic larvae 25%, terrestrial insects 14%, aquatic insects 7% and crustacea 5%. Juvenile S. intermedius feed predominantly on insect larvae, while adults are largely piscivorous.     

Oviposition‐induced plant cues: do they arrest Trichogramma wasps during host location?

Plants can defend themselves against herbivorous insects before the larvae hatch from eggs and start feeding. One of these preventive defence strategies is to produce plant volatiles, in response to egg deposition, which attract egg parasitoids that subsequently kill the herbivore eggs. Here, we studied whether egg deposition by Pieris brassicae L. (Lepidoptera: Pieridae) induces Brussels sprouts plants to produce cues that attract or arrest Trichogramma brassicae Bezdeko (Hymenoptera: Trichogrammatidae). Olfactometer bioassays revealed that odours from plants with eggs did not attract or arrest parasitoids. However, contact bioassays showed that T. brassicae females were arrested on egg‐free leaf squares excised from leaves with 72 h‐old egg masses, which are highly suitable for parasitisation. We tested the hypothesis that this arresting activity is due to scales and chemicals deposited by the butterflies during oviposition and which are thus present on the leaf surface in the vicinity of the eggs. Indeed, leaf squares excised from egg‐free leaves, but contaminated with butterfly deposits, arrested the wasps when the squares were tested 1 day after contamination. However, squares from egg‐free leaves with 72 h‐old butterfly deposits had no arresting activity. Thus, we exclude that the arresting activity of the leaf area near 72 h‐old egg masses was elicited by cues from scales and other butterfly deposits. We suggest that egg deposition of P. brassicae induces a change in the leaf surface chemicals in leaves with egg masses. A systemic induction extending to an egg‐free leaf neighbouring an egg‐carrying leaf could not be detected. Our data suggest that a local, oviposition‐induced change of leaf surface chemicals arrests T. brassicae in the vicinity of host eggs.     

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.     

Egg maturation dynamics of the parasitoid Microplitis rufiventris: starvation speeds maturation in early life

The number of mature eggs carried by a female parasitoid at any given moment (egg load) is a fitness‐related parameter affecting reproductive potential and impacting upon host population dynamics. Microplitis rufiventris Kokujev (Hymenoptera: Braconidae) is a solitary koinobiont endoparasitoid wasp of several noctuid pests, including Spodoptera littoralis. The number of mature eggs carried by females at emergence is approximately 50. The rate of egg maturation is strongly affected both by feeding status and access to host larvae. In early adult life, egg maturation rates are lower for 6–72 h in fed wasps compared with food‐deprived wasps. When given access to hosts, honey‐fed wasps live for approximately 9 days with high lifetime fecundity (226 eggs). By contrast to early adult life, the total realized fecundity is positively affected by feeding status, where water‐fed and starved females have 140 and 107 eggs, respectively. Egg resorption is most pronounced in the later life of females. The results suggest, in addition to confirming the effect of honey‐feeding on total fecundity, that fecundity of starved wasps includes rapid egg maturation early in life, which potentially could improve the performance of the parasitoid as a biological control agent.     

High genetic diversity of Vibrio cholerae in the European lake Neusiedler See is associated with intensive recombination in the reed habitat and the long‐distance transfer of strains

Coastal marine Vibrio cholerae populations usually exhibit high genetic diversity. To assess the genetic diversity of abundant V. cholerae non‐O1/non‐O139 populations in the Central European lake Neusiedler See, we performed a phylogenetic analysis based on recA, toxR, gyrB and pyrH loci sequenced for 472 strains. The strains were isolated from three ecologically different habitats in a lake that is a hot‐spot of migrating birds and an important bathing water. We also analyzed 76 environmental and human V. cholerae non‐O1/non‐O139 isolates from Austria and other European countries and added sequences of seven genome‐sequenced strains. Phylogenetic analysis showed that the lake supports a unique endemic diversity of V. cholerae that is particularly rich in the reed stand. Phylogenetic trees revealed that many V. cholerae isolates from European countries were genetically related to the strains present in the lake belonging to statistically supported monophyletic clades. We hypothesize that the observed phenomena can be explained by the high degree of genetic recombination that is particularly intensive in the reed stand, acting along with the long distance transfer of strains most probably via birds and/or humans. Thus, the Neusiedler See may serve as a bioreactor for the appearance of new strains with new (pathogenic) properties.     

Predator diversity effects cascade across an ecosystem boundary

Food webs in different ecosystems are often connected through spatial resource subsidies. As a result, biodiversity effects in one ecosystem may cascade to adjacent ecosystems. I tested the hypothesis that aquatic predator diversity effects cascade to terrestrial food webs by altering a prey subsidy (biomass and trophic structure of emerging aquatic insects) entering terrestrial food webs, in turn altering the distribution of a terrestrial consumer (spider) that feeds on emerging aquatic insects. Fish presence, but not diversity, altered the trophic structure of emerging aquatic insects by strongly reducing the biomass of emerging predators (dragonflies) relative to non‐feeding taxa (chironomid midges). Fish diversity reduced emerging insect biomass through enhanced effects on the most common prey taxa: predatory dragonflies Pantala flavescens and non‐feeding chironomids. Terrestrial spiders (Tetragnathidae) primarily captured emerging chironomids, which were reduced in the high richness (3 spp.) treatment relative to the 1 and 2 species treatments. As a result, terrestrial spider abundance was lower above pools with high fish richness (3 species) than pools with 1 and 2 species. Synergistic predation effects were mostly limited to the high richness treatment, in which fish occupied each level of vertical microhabitat in the water‐column (benthic, middle, surface). This study demonstrates that predator diversity effects are not limited to the habitat of the predator, but can propagate to adjacent ecosystems, and demonstrates the utility of using simple predator functional traits (foraging domain) to more accurately predict the direction of predator diversity effects.     

Does mother know best? The preference–performance hypothesis and parent–offspring conflict in aboveground–belowground herbivore life cycles

1. A substantial amount of research on host‐plant selection by insect herbivores is focused around the preference–performance hypothesis (PPH). To date, the majority of studies have primarily considered insects with aboveground life cycles, overlooking insect herbivores that have both aboveground and belowground life stages, for which the PPH could be equally applicable. 2. This study investigated the factors influencing the performance of the root‐feeding vine weevil (Otiorhynchus sulcatus) larvae and whether this was linked to the oviposition behaviour of the maternal adult living aboveground. 3. Maternal insects feeding aboveground reduced root biomass by 34% and increased root carbon by 4%. Larvae feeding on plants subjected to aboveground herbivory had reduced mass. Irrespective of the presence of maternal herbivory, larval mass was positively correlated with root biomass. 4. Larval mass was also reduced by conspecific larvae, previously feeding on roots (19% reduction). However, the mechanism underpinning this effect remains unclear, as in contrast to maternal herbivory aboveground, prior larval feeding did not significantly affect root biomass or root carbon concentrations. 5. Maternal insects did not distinguish between plants infested with larvae and those that were free of larvae, in terms of their egg‐laying behaviour. Conversely, maternal insects tended to lay eggs on plants with smaller root systems, a behaviour that is likely to negatively affect offspring performance. 6. The PPH is not supported by our findings for the polyphagous vine weevil feeding on the host plant raspberry (Rubus idaeus), and in fact our results suggest that there is the potential for strong parent–offspring conflict in this system.     

Transfer of cholera toxin genes from O1 to non‐O1/O139 strains by vibriophages from California coastal waters

Aims: Vibrio cholerae is an important bacterial pathogen that causes global cholera epidemic. Although they are commonly found in coastal waters around the world, most environmental isolates do not contain cholera toxin genes. This study investigates vibriophages in southern California coastal waters and their ability to transfer cholera toxin genes. Methods and Results: Lytic phages infecting V. cholerae were isolated from Newport Bay, California, between May and November, while none was found in winter. Some of the phage isolates can infect multiple environmental V. cholerae strains and El Tor strains. All phages contained double‐stranded DNA. Transduction experiments using kanamycin‐resistant gene marked CTXΦ demonstrated that some environmental vibriophages can transfer CTXΦ genes from O1 El Tor strain to environmental non‐O1/O139 V. cholerae via generalized transduction. Conclusions: Vibriophages are important components of the natural aquatic ecosystem. They play an important role in influencing the dynamics and evolution of V. cholerae in the environment. Significance and Impact of the Study: This study demonstrates the significance of vibriophages in the coastal environment and transduction as one of the mechanisms of pathogenicity evolution among environmental V. cholerae.     

Predation risk affects egg mortality and carry over effects in the larval stages in damselflies

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Caddisfly egg mass morphology mediates egg predation: potential costs to individuals and populations

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The roles of history: age and prior exploitation in aquatic container habitats have immediate and carry‐over effects on mosquito life history

1. Per‐capita resource availability in aquatic habitats is influenced directly by consumer density via resource competition and indirectly via delayed resource competition when temporally non‐overlapping cohorts of larvae exploit the same resources. In detritus‐based systems, resources are likely to be influenced by the age of the aquatic habitat, as detritus changes in quality over time and may be replenished by new inputs. 2. For aquatic insects that exploit detritus‐based habitats, feeding conditions experienced during immature stages can influence fitness directly via effects on development and survivorship, but also indirectly by influencing adult traits such as fecundity and longevity. 3. Larval habitat age and prior resource exploitation were manipulated in a field experiment using the container mosquito Aedes triseriatus. 4. It was found that A. triseriatus from older habitats had greater larval survival, faster development and greater adult longevity. Exploitation of larval habitats by a prior cohort of larvae had a significant negative effect on subsequent cohorts of larvae by delaying development. 5. It is suggested that extended conditioning of detritus probably resulted in conversion of recalcitrant resources to more available forms which improved the quality of the habitat. 6. In a parallel study, evidence was found of carry‐over effects of habitat age and prior exploitation on adult longevity for A. triseriatus and Aedes japonicus collected from unmanipulated aquatic habitats. 7. These results indicate the importance of detritus dynamics and the discontinuous nature of resource competition in these mosquito‐dominated aquatic systems.