Multiple symbiotes in larval black flies (Diptera: Simuliidae): associations of trichomycete fungi with ichthyosporeans, mermithid nematodes, and microsporidia

Studies of the interactions between two or more species of symbiotic microorganisms in a single host animal have lagged behind those involving interactions between hymenopteran or dipteran parasitoids. We screened the larvae of four species of black flies (Cnephia ornithophilia, Simulium furculatum, Simulium innoxium, and Simulium vandalicum) to determine if the colonization and prevalence of trichomycete fungi were influenced by ichthyosporeans, mermithid nematodes, microsporidia, or other species of trichomycetes. Differences in the layering of the peritrophic matrix of microsporidian-infected larvae, compared with that of uninfected larvae, were associated with greater levels of colonization by the midgut-inhabiting trichomycete Harpella melusinae. Colonization levels of different species of trichomycetes were significantly correlated with one another in only 2 of 11 cases. Aside from the consistent attachment of the trichomycete Simuliomyces microsporus to the ichthyosporean Paramoebidium chattoni, we found little evidence that prevalence and colonization levels of trichomycetes were influenced by other symbiotes, including other trichomycetes. This trend might reflect the physical separation of trichomycetes, which are gut specific, from other symbiotes. In addition, any combinations of symbiotes that were wholly incompatible would not have been observed in our field-collected material.     

First report of a trichomycete fungus (Zygomycota: Trichomycetes) inhabiting larvae of Simulium ochraceum sensu lato Walker (Diptera: Simuliidae) from the Galapagos Islands

A collection of the larval black fly Simulium ochraceum sensu lato Walker was made at six lotic habitats on San Cristobal Island, Galapagos Islands, Ecuador, during May 2003. Sixty-eight larvae were assayed for the presence of gut fungi (Zygomycota: Trichomycetes). A trichomycete, designated as Smittium sp. was found inhabiting the hindgut of 17.6% of S. ochraceum s.l. larvae. Implications of this finding with respect to both host and trichomycete biogeography are discussed.     

Seasonality of trichomycetes in larval black flies from South Carolina, USA

Trichomycete fungi are common endobionts of aquatic insect larvae, but little is known of their ecology. In this study, the seasonality of trichomycete colonization of larval black flies (Diptera: Simuliidae) was investigated in three streams in northwestern South Carolina. At least eight species of trichomycetes were found in two species of black flies, and 93.8% of 1819 larval black flies examined contained trichomycetes. Significant differences were found in the seasonal prevalence of Harpella melusinae, Simuliomyces microsporus, and Paramoebidium spp. at one of three sites. At this site, the lowest mean prevalence for H. melusinae occurred in winter (67%) versus the other seasons (96-100%), whereas mean prevalence was lowest in summer for Simuliomyces microsporus (1%) versus the other seasons (2-21%) and lowest in summer for P. spp. (9%) versus the other seasons (45-67%). Significant differences in levels of colonization among seasons were not detected. Conjugations of H. melusinae (representing early stages of sexual reproduction) occurred most frequently in the spring and winter (up to 14% of larvae). Sexual reproduction (represented by zygospores) of Legeriomycetaceae occurred most frequently in the spring and fall (up to 17% of larvae).     

Do gut symbiotes reflect the endemism of their host black flies (Diptera: Simuliidae) in the Caucasus of Armenia?

Aim We tested the hypothesis that endemism of black flies in the Caucasus of Armenia is reflected in their gut symbiotes, i.e. trichomycete fungi and ichthyosporean protists. Location The study area, Armenia, is a biodiversity hotspot, with high levels of plant and animal endemism. Located in the southern Caucasus, Armenia is a rugged, mountainous, landlocked country of 29,800 km², with fast‐flowing rivers and little forested land. About half of the 51 species of black flies in Armenia are endemic to the Caucasus. Methods The larvae of 22 species of black flies, including seven endemic to the Caucasus, were collected from 35 sites throughout Armenia, from 1998 to 2004, and assayed microscopically for gut‐inhabiting trichomycete fungi and ichthyosporean protists. Results Nearly 68% of larval black flies in Armenia contained at least one species of gut symbiote, with a mean of 0.9 ± 0.04 species per host larva. Eight species of trichomycete fungi and two species of ichthyosporeans were found in larval guts. Seven of these species were recorded for the first time in Asian black flies. Species accumulation curves showed no additional species of gut symbiotes after 17 host species and 22 sites had been examined. All gut symbiotes were widely distributed, indicating an absence of endemic trichomycetes in the black flies of Armenia. Main conclusions Although the Caucasus Region is noted for its high levels of endemism for plants and animals, including black flies, the gut symbiotes of black flies are all widespread species. The absence of endemic gut symbiotes in larval black flies might be explained by their lack of host specificity; an absence of host specificity would not constrain the geographical distribution of the symbiotes.     

Susceptibility of black soldier fly (Diptera: Stratiomyidae) larvae and adults to four insecticides

Dosage-mortality regressions were determined for black soldier fly, Hermetia illucens (L.), larvae fed cyromazine or pyriproxifen treated media. Cyromazine LC50 for larvae dying before becoming prepupae ranged from 0.25 to 0.28 ppm with dosage-mortality regression slopes between 5.79 and 12.04. Cyromazine LC50s for larvae dying before emergence ranged from 0.13 to 0.19 ppm with dosage-mortality regression slopes between 3.94 and 7.69. Pyriproxifen dosage-mortality regressions were not generated for larvae failing to become prepupae since <32% mortality was recorded at the highest concentration of 1,857 ppm. LC50s for larvae failing to become adults ranged from 0.10 to 0.12 ppm with dosage mortality-regression slopes between 1.67 and 2.32. Lambda-cyhalothrin and permethrin dosage-mortality regressions were determined for wild adult black soldier flies and house flies, Musca domestica L., and for susceptible house flies. Our results indicate that the wild house fly, unlike the black soldier fly, population was highly resistant to each of these pyrethroids. Regression slopes for black soldier flies exposed to lambda-cyhalothrin were twice as steep as those determined for the wild house fly strain. Accordingly, LC50s for the black soldier fly and susceptible house fly were 10- to 30-fold lower than those determined for wild house flies. The differential sensitivity between wild black soldier flies and house flies might be due to behavioral differences. Adult house flies usually remain in animal facilities with the possibility of every adult receiving pesticide exposure, while black soldier fly adults are typically present only during emergence and oviposition thereby limiting their exposure.     

Oviposition by Aedes aegypti (Diptera: Culicidae) in relation to conspecific larvae infected with internal symbiotes.

Female mosquitoes use abiotic and biotic factors, such as the presence of heavy metals or parasites, to determine the acceptability of oviposition sites. Some biotic factors can originate from mosquito larvae. When mosquito larvae are starved or infected with trematode metacercaria, they render associated water unacceptable to ovipositioning females. Internal symbiotes are common in wild mosquito populations, and I tested whether or not larvae of Aedes aegypti infected with a gregarine (Ascogregarina taiwanensis), a yeast (Candida near pseudoglaebosa), or a trichomycete (Smittium morbosum) rendered their rearing water unacceptable to oviposting mosquitoes. Infections with S. morbosum had no effect on the acceptability of the associated rearing water when compared to rearing water from uninfected larvae. However, the rearing water from larvae infected with A. taiwanensis or C. near pseudoglaebosa was more acceptable to ovipositing females than was distilled water or rearing water from uninfected larvae. Ovipositing female mosquitoes either preferred or were neutral to rearing water from larvae with gut symbiotes.     

Inoculating poultry manure with companion bacteria influences growth and development of black soldier fly (Diptera: Stratiomyidae) larvae

The growth and development of black soldier fly, Hermetia illucens (L.), larvae fed chicken manure inoculated with bacteria isolated from black soldier fly larvae and associated larval feed was evaluated. Four strains of Bacillus subtilis were evaluated. B. subtilis strains S15, S16, S19, were isolated from the gut of black soldier fly larvae. B. natto strain D1 was isolated from the diet fed to black soldier fly larvae. These bacteria were added individually into nonsterile 200 g fresh hen manure at 10(6) cfu/g and homogenized. Treated manure was then inoculated with 4-d old black soldier fly larvae. Prepupal weight ranged from 0.0606 g in the control to 0.0946 g in manure treated with the S15 strain. Larval survivorship to the prepupal stage in all treatments ranged from 98.00 ± 2.65% to 99.33 ± 1.15%. Prepupal survivorship to the pupal stage ranged from 91.92 ± 1.87% to 97.95 ± 1.03%. Adult emergence from the pupal stage did not significantly (P < 0.05) differ across treatments and ranged from 98.95 ± 1.82% to 100.00 ± 0.00%. Adult body length resulting from the larvae in each of the treatments was significantly greater than those from the control. Longevity of adults did not differ significantly between treatments. Time from hatching to the development of the first pupa did not differ significantly across treatments; however, development time from hatching to 90% reaching the prepupual stage was significantly different between treatments and ranged from 29.00 ± 1.00 d to 34.33 ± 3.51 d. Development time from hatching to 90% reaching the adult stages was significantly different between treatments. Our results demonstrate that inoculating poultry manure with bacteria from black soldier fly larvae influences the growth and development of conspecific larvae feeding on the manure.     

Biochemical characterization of digestive enzymes in the black soldier fly,Hermetia illucens (Diptera: Stratiomyidae)

The black soldier fly, Hermetia illucens, is beneficial because its larvae feed on organic materials derived from plants, animals and humans and promote the recycling of food waste and organic materials. We investigated the biochemical properties of digestive enzymes released from the salivary gland and gut of the black soldier fly. Because the gut extracts of the black soldier fly larvae had high amylase, lipase and protease activities, we suggested that the black soldier fly might belong to the polyphagous insect group. In addition, a strong trypsin-like protease activity was observed in the gut extracts of the black soldier fly larvae. Higher activities of leucine arylamidase, α-galactosidase, β-galactosidase, α-mannosidase and α-fucosidase were observed from the gut extracts of the black soldier fly larvae compared with those of house fly larvae. These findings may explain previous reports that the black soldier fly larvae can digest food wastes and organic materials more efficiently than any other known species of fly.     

Characteristics of pathogenic and mutualistic relationships of ascoviruses in field populations of parasitoid wasps

Ascoviruses are disseminated among larvae in lepidopteran populations by parasitic wasps during oviposition. Ascovirus relationships with these wasps vary from pathogenic to mutualistic, and experimentally can be shown possibly to be commensal non-pathogenic virus having little or no effect. Most ascoviruses are pathogens that female wasps vector mechanically. Other ascoviruses have a more intimate relationship with their wasp vectors in that their genome is stably maintained in all wasp nuclei through several generations by vertical transmission. In this relationship, these viruses are mutualistic, enhancing the successful development of the wasp larvae by suppressing lepidopteran defence mechanisms. The DpAV4 ascovirus is a mutualist in certain Diadromus wasps but is pathogenic or not when vectored by other species of this genus. These various biologies suggest that ascovirus/wasp relationships depend on wasp regulatory factors that control virus replication. Thus, certain ascoviruses can potentially have either a pathogenic, mutualistic, or non-pathogenic relationship with a specific wasp vector, the type of relationship being dependent upon the species system in which the relationship evolved. Finally, because ascoviruses appear to be related to ichnoviruses (Polydnaviridae), the DpAV4/Diadromus system constitutes a possible interesting intermediate between the pathogenic ascoviruses and symbiotic viruses that evolved to be ichnoviruses.     

The costs and benefits in an unusual symbiosis: experimental evidence that bitterling fish (Rhodeus sericeus) are parasites of unionid mussels in Europe

Interspecific symbiotic relationships involve a complex network of interactions, and understanding their outcome requires quantification of the costs and benefits to both partners. We experimentally investigated the costs and benefits in the relationship between European bitterling fish (Rhodeus sericeus) and freshwater mussels that are used by R. sericeus for oviposition. This relationship has hitherto been thought mutualistic, on the premise that R. sericeus use mussels as foster parents of their embryos while mussels use R. sericeus as hosts for their larvae. We demonstrate that R. sericeus is a parasite of European mussels, because it (i) avoids the cost of infection by mussel larvae and (ii) imposes a direct cost on mussels. Our experiments also indicate a potential coevolutionary arms race between bitterling fishes and their mussel hosts; the outcome of this relationship may differ between Asia, the centre of distribution of bitterling fishes, and Europe where they have recently invaded.     

Starvation period and age affect the response of female Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) to odor and visual cues

The effects of starvation or age on the walking or flying response of female Frankliniella occidentalis to visual and/or odor cues in two types of olfactometer were examined in the laboratory. The response of walking thrips starved for 0, 1, 4, or 24h to an odor cue (1microl of 10% p-anisaldehyde) was examined in a Y-tube olfactometer. The take-off and landing response of thrips (unknown age) starved for 0, 1, 4, 24, 48 or 72h, or of thrips of different ages (2-3 days or 10-13 days post-adult emergence) starved for 24h, to a visual cue (98 cm(2) yellow sticky trap) and/or an odor cue (0.5 or 1.0 ml p-anisaldehyde) was examined in a wind tunnel. More thrips walked up the odor-laden arm in the Y-tube when starved for at least 4h (76%) than satiated thrips (58.7%) or those starved for 1h (62.7%, P<0.05). In the wind tunnel experiments the percentage of thrips to fly or land on the sticky trap increased between satiated thrips (7.3% to fly, 3.3% on trap) and those starved for 4h (81.2% to fly, 29% on trap) and decreased between thrips starved for 48 (74.5% to fly, 23% on trap) and 72 h (56.5% to fly, 15.5% on trap, P<0.05). Fewer younger thrips (38.8%) landed on a sticky trap containing a yellow visual cue of, those that flew, than older thrips (70.4%, P<0.05), although a similar percentage of thrips flew regardless of age or type of cue present in the wind tunnel (average 44%, P>0.05).     

黑水虻幼虫蛋白及其酶解产物的抗氧化活性研究

为了探究黑水虻Hermetia illucens幼虫蛋白及酶解产物的抗氧化活性,以鲜活黑水虻幼虫冻虫为原料,采用碱提酸沉法提取黑水虻蛋白,通过碱性蛋白酶、菠萝蛋白酶、风味蛋白酶、木瓜蛋白酶对其蛋白质溶液进行酶解,分别从2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸（ABTS）自由基、羟自由基、1,1-二苯基-2-三硝基苯肼（DPPH）自由基3个方面对黑水虻蛋白及其酶解液的抗氧化能力进行测定。结果表明：黑水虻幼虫蛋白及4种酶解后的蛋白肽具有较好的抗氧化活性,其中黑水虻幼虫蛋白对ABTS自由基、羟自由基、DPPH自由基的半抑制浓度（IC50）分别为2.91、0.232、0.764 mg/mL,而酶解过后的蛋白肽具有更强的抗氧化活性,对ABTS自由基、羟自由基、DPPH自由基的最低半抑制浓度（IC50）分别为0.295、0.082、0.354 mg/mL。本研究初步证明了酶解制备抗氧化肽的可行性,为昆虫蛋白资源的利用和无抗饲料的研制提供了新的研究思路。     

Prevalence of the trichomycete fungus Harpella melusinae (Harpellales: Harpellaceae) in larval black flies (Diptera: Simuliidae) across a heterogeneous environment

A total of 2063 mid- to late-instar larval black flies were collected from 64 stream sites in South Carolina and screened for the presence of the trichomycete fungus Harpella melusinae. Sixteen of 18 host species were colonized by H. melusinae on at least one occasion. Prevalence of H. melusinae in larvae of Simulium tuberosum cytospecies "A" was highest in acidic streams with low conductivity, whereas H. melusinae colonized larvae of Simulium verecundum most frequently in slower-moving streams. Ecological conditions, therefore, can serve as predictors of the prevalence of H. melusinae. Prevalence in host larvae was significantly lower in the Piedmont ecoregion than in the Mountain ecoregion. Prevalence did not differ in the host species S. verecundum across ecoregions, suggesting that different prevalences among host species might indicate some host preference. The prevalence of H. melusinae differed significantly between two univoltine host species (Simulium venustum and Prosimulium magnum) at the same site but not between two multivoltine host species (S. tuberosum cytospecies "FG" and S. tuberosum cytospecies "CDE"), suggesting that host life history could be important in determining fungal prevalence.     

Gregarines on a diet: the effects of host starvation on Gregarina confusa Janovy et al., 2007 (Apicomplexa: Eugregarinida) in Tribolium destructor Uyttenboogaart, 1933 (Coleoptera: Tenebrionidae) larvae

This study was designed to explore the nutritional relationship between Gregarina confusa (Apicomplexa: Eugregarinida) parasites and its coleopteran host, Tribolium destructor, by measuring the cytoplasmic density of gregarines in continuously fed larvae, starved larvae, and larvae refed after starvation. Cultures were maintained in a standard media (whole wheat flour:commercial wheat germ:yeast [30:10:1]). Larvae from control and experimental groups were dissected daily for 3 days then allowed to feed or starve for an additional 3 days. On day 6, the remaining experimental larvae were divided and placed into 2 groups; 1 group remained starved while larvae from the second group were fed a Wheaties flake. Photographs were taken of the parasites daily and analyzed using ScionImage. Gregarines from starved larvae were significantly longer and skinnier than those from fed controls, and there was also a significant difference between gregarine deutomerite cytoplasmic densities. Parasites from refed larvae regained cytoplasmic density within 24 hr and showed morphological similarities to those from fed larvae. This study shows that the Tribolium destructor-Gregarina confusa relationship can be manipulated easily through alterations of host diet and thus is an excellent model for use in the study of chemical relationships between parasites and their hosts.     

Black soldier fly (Diptera: Stratiomyidae) larval heat generation and management

Mass production of black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), larvae results in massive heat generation, which impacts facility management, waste conversion, and larval production. We tested daily substrate temperatures with different population densities (i.e., 0, 500, 1000, 5000, and 10 000 larvae/pan), different population sizes (i.e., 166, 1000, and 10 000 larvae at a fixed feed ratio) and air temperatures (i.e., 20 and 30 °C) on various production parameters. Impacts of shifting larvae from 30 to 20 °C on either day 9 or 11 were also determined. Larval activity increased substrate temperatures significantly (i.e., at least 10 °C above air temperatures). Low air temperature favored growth with the higher population sizes while high temperature favored growth with low population sizes. The greatest average individual larval weights (e.g., 0.126 and 0.124 g) and feed conversion ratios (e.g., 1.92 and 2.08 g/g) were recorded for either 10 000 larvae reared at 20 °C or 100 larvae reared at 30 °C. Shifting temperatures from high (30 °C) to low (20 °C) in between (∼10-d-old larvae) impacted larval production weights (16% increases) and feed conversion ratios (increased 14%). Facilities should consider the impact of larval density, population size, and air temperature during black soldier fly mass production as these factors impact overall larval production.     

Development of black soldier fly (Diptera: Stratiomyidae) larvae fed dairy manure

Black soldier flies, Hermetia illucens L., are a common colonizer of animal wastes. However, all published development data for this species are from studies using artificial diets. This study represents the first examining black soldier fly development on animal wastes. Additionally, this study examined the ability of black soldier fly larvae to reduce dry matter and associated nutrients in manure. Black soldier fly larvae were fed four rates of dairy manure to determine their effects on larval and adult life history traits. Feed rate affected larval and adult development. Those fed less ration daily weighed less than those fed a greater ration. Additionally, larvae provided the least amount of dairy manure took longer to develop to the prepupal stage; however, they needed less time to reach the adult stage. Adults resulting from larvae provided 27 g dairy manure/d lived 3-4 d less than those fed 70 g dairy manure. Percentage survivorship to the prepupal or adult stages did not differ across treatments. Larvae fed 27 g dairy manure daily reduced manure dry matter mass by 58%, whereas those fed 70 g daily reduced dry matter 33%. Black soldier fly larvae were able to reduce available P by 61-70% and N by 30-50% across treatments. Based on results from this study, the black soldier fly could be used to reduce wastes and associated nutrients in confined bovine facilities.     

Geographical and within-population variation in the globeflower–globeflower fly interaction: the costs and benefits of rearing pollinator’s larvae

Interspecific interactions can vary within and among populations and geographical locations, and this variation can influence the nature of the interaction (e.g. mutualistic versus antagonistic) and its evolutionary stability. Globeflowers are exclusively pollinated by flies whose larvae feed only on their seeds. Here we document geographical variability in costs and benefits in globeflowers in sustaining their pollinating flies throughout the range of this arctic-alpine European plant over several years. A total of 1,710 flower heads from 38 populations were analysed for their carpel, egg and seed contents. Individual and population analyses control for the confounding influences of variation in both: (1) population traits, such as fly density and egg distribution among flower heads; and (2) individuals traits, such as carpel and egg numbers per flower head. Despite considerable variation in ecological conditions and pollinator densities across populations, large proportions (range 33–58%) of seeds are released after predation, with a benefit-to-cost ratio of 3, indicating that the mutualism is stable over the whole globeflower geographical range. The stability of the mutualistic interaction relies on density-dependent competition among larvae co-developing in a flower head. This competition is revealed by a sharp decrease in the number of seeds eaten per larva with increasing larval number, and is intensified by non-uniform egg distribution among globeflowers within a population. Carpel number is highly variable across globeflowers (range 10–69), and flies lay more eggs in large flowers. Most plants within a population contribute to the rearing of pollinators, but some pay more than others. Large globeflowers lose more seed to pollinator larvae, but also release more seed than smaller plants. The apparent alignment of interests between fly and plant (positive relationship between numbers of seeds released and destroyed) is shown to hide a conflict of interest found when flower size is controlled for.     

Geographic and within-population variation in the globeflower–globeflower fly interaction: the costs and benefits of rearing pollinators’ larvae

Interspecific interactions can vary within and among populations and geographic locations, and this variation can influence the nature of the interaction (e.g. mutualistic vs. antagonistic) and its evolutionary stability. Globeflowers are exclusively pollinated by flies, whose larvae feed only on their seeds. Here we document geographic variability in costs and benefits in globeflowers in sustaining their pollinating flies throughout the range of this arctic-alpine European plant over several years. A total of 1,710 flower heads from 38 populations were analysed for their carpel, egg and seed contents. Individual and population analyses control for the confounding influences of variation in both: (1) population traits, such as fly density and egg distribution among flower heads; and (2) individuals traits, such as carpel and egg numbers per flower head. Despite considerable variation in ecological conditions and pollinator densities across populations, large proportions (range 33–58%) of seeds were released after predation, with a benefit-to-cost ratio of 3, indicating that the mutualism is stable over the whole globeflower geographical range. The stability of the mutualistic interaction relies on density-dependent competition among larvae co-developing in a flower head. This competition is revealed by a sharp decrease in the number of seeds eaten per larva with increasing larval number, and is intensified by non-uniform egg distribution among globeflowers within a population. Carpel number is highly variable across globeflowers (range 10–69), and flies lay more eggs in large flowers. Most plants within a population contribute to the rearing of pollinators, but the costs are greater for some than for others. Large globeflowers lose more seed to pollinator larvae, but also release more seed than smaller plants. The apparent alignment of interests between fly and plants (positive relationship between numbers of seed released and destroyed) is shown to hide a conflict of interest found when flower size is controlled for.     

Field response of sugarcane cultivars to sugarcane soldier fly, Inopus rubriceps (Macquart) (Diptera: Stratiomyidae)

Abstract Ten commercial sugarcane cultivars were planted in 10 replicate plots in each of three field trials in southern Queensland, to evaluate their response to the sugarcane soldier fly Inopus rubriceps . Trials were monitored for three harvests. Numbers of soldier fly larvae differed slightly between cultivars, but probably not enough to prevent the development of damaging populations. Weights of larvae were the same on all cultivars, suggesting that antibiosis was not involved. The effect of soldier fly on performance of cultivars in each trial was examined by regression of number of ratoon shoots and subsequent number and weight of mature stalks and crop yield on larval density during summer−autumn within each crop class. Where statistically significant relationships were detected in ratoons, each increase in soldier fly density of 100 m⁻² was associated with a yield reduction of 1.2−6.4 tonnes cane ha⁻¹, or 3−8% of the expected yield of uninfested plants. In most analyses, the slopes of regressions were the same for all cultivars, indicating no differential response to soldier fly infestation among the cultivars tested.