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.     

Hostplant,larval age,and feeding behavior influence midgut pH in the gypsy moth (Lymantria dispar)

Summary The midgut pH of late instar gypsy moth (Lymantria dispar L.) larvae is strongly alkaline, and varies with diet, larval stadium, and time since feeding. Midgut pH rises with time since feeding, and does so more quickly, reaching greater maximum values, on some diets than others. Leaf tissues of 23 tree species resist increases in alkalinity differentially; this trait and differing initial leaf pH may explain the impact of diet on gut pH. Third instar larvae may have gut conditions favorable for tannin-protein binding shortly after ingesting certain foods, but with time midgut alkalinity becomes great enough to dissociate tannin-protein complexes. Older instars rarely exhibit gut pHs low enough to permit tannin activity. Alkaline gut conditions may explain the gypsy moth's ability to feed on many tanniniferous plant species, especially in later instars. Consequences for pathogen effectiveness are discussed.     

Gut microbiota of dung beetles correspond to dietary specializations of adults and larvae

Vertebrate dung is central to the dung beetle life cycle, constituting food for adults and a protective and nutritive refuge for their offspring. Adult dung beetles have soft mandibles and feed primarily on nutritionally rich dung particles, while larvae have sclerotized mandibles and consume coarser dung particles with a higher C/N ratio. Here, using the dung beetles Euoniticellus intermedius and E. triangulatus, we show that these morphological adaptations in mandibular structure are also correlated with differences in basic gut structure and gut bacterial communities between dung beetle life stages. Metagenome functional predictions based on 16S rDNA characterization further indicated that larval gut communities are enriched in genes involved in cellulose degradation and nitrogen fixation compared to adult guts. Larval gut communities are more similar to female gut communities than they are to those of males, and bacteria present in maternally provisioned brood balls and maternal ‘gifts’ (secretions deposited in the brood ball along with the egg) are also more similar to larval gut communities than to those of males. Maternal secretions and maternally provisioned brood balls, as well as dung, were important factors shaping the larval gut community. Differences between gut microbiota in the adults and larvae are likely to contribute to differences in nutrient assimilation from ingested dung at different life history stages.     

Similarity and specialization of the larval versus adult diet of European butterflies and moths

Many herbivorous insects feed on plant tissues as larvae but use other resources as adults. Adult nectar feeding is an important component of the diet of many adult herbivores, but few studies have compared adult and larval feeding for broad groups of insects. We compiled a data set of larval host use and adult nectar sources for 995 butterfly and moth species (Lepidoptera) in central Europe. Using a phylogenetic generalized least squares approach, we found that those Lepidoptera that fed on a wide range of plant species as larvae were also nectar feeding on a wide range of plant species as adults. Lepidoptera that lack functional mouthparts as adults used more plant species as larval hosts, on average, than did Lepidoptera with adult mouthparts. We found that 54% of Lepidoptera include their larval host as a nectar source. By creating null models that described the similarity between larval and adult nectar sources, we furthermore showed that Lepidoptera nectar feed on their larval host more than would be expected if they fed at random on available nectar sources. Despite nutritional differences between plant tissue and nectar, we show that there are similarities between adult and larval feeding in Lepidoptera. This suggests that either behavioral or digestive constraints are retained throughout the life cycle of holometabolous herbivores, which affects host breadth and identity.     

The Intestinal Bacterial Community in the Food Waste-Reducing Larvae of <Emphasis Type="Italic">Hermetia illucens</Emphasis>

As it is known that food waste can be reduced by the larvae of Hermetia illucens (Black soldier fly, BSF), the scientific and commercial value of BSF larvae has increased recently. We hypothesised that the ability of catabolic degradation by BSF larvae might be due to intestinal microorganisms. Herein, we analysed the bacterial communities in the gut of BSF larvae by pyrosequencing of extracting intestinal metagenomic DNA from larvae that had been fed three different diets. The 16S rRNA sequencing results produced 9737, 9723 and 5985 PCR products from larval samples fed food waste, cooked rice and calf forage, respectively. A BLAST search using the EzTaxon program showed that the bacterial community in the gut of larvae fed three different diets was mainly composed of the four phyla with dissimilar proportions. Although the composition of the bacterial communities depended on the different nutrient sources, the identified bacterial strains in the gut of BSF larvae represented unique bacterial species that were unlike the intestinal microflora of other insects. Thus, our study analysed the structure of the bacterial communities in the gut of BSF larvae after three different feedings and assessed the application of particular bacteria for the efficient degradation of organic compounds.     

Evolutionary changes in the timing of gut morphogenesis in larvae of the marine annelid Streblospio benedicti

The planktonic larvae of marine invertebrates are diverse in their nutritional modes, suggesting that evolutionary transitions in larval nutritional mode have been frequent. One approach to identifying the developmental changes that play important roles in such transitions is to compare "intermediate" larval forms to closely related larvae representative of their common ancestor. Here we make such a comparison between obligately planktotrophic and facultatively feeding larvae of the poecilogonous polychaete annelid Streblospio benedicti. We used feeding experiments to show that the derived, facultatively feeding larvae of this species develop the ability to feed at a later developmental stage (five muscle bands) than planktotrophic larvae (two to three muscle bands). This delay in the onset of feeding ability does not appear to be caused by delay in the formation of particle capture structures, but instead by delay in the development of a continuous, functional gut. These observations are consistent with the hypothesis that evolutionary increases in egg size in annelids lead predictably to heterochronic delays in gut development, and hence to transitions in larval nutritional mode.     

Spatial aggregation across ephemeral resource patches in insect communities: an adaptive response to natural enemies?

Although an increase in competition is a common cost associated with intraspecific crowding, spatial aggregation across food-limited resource patches is a widespread phenomenon in many insect communities. Because intraspecific aggregation of competing insect larvae across, e.g. fruits, dung, mushrooms etc., is an important means by which many species can coexist (aggregation model of species coexistence), there is a strong need to explore the mechanisms that contribute to the maintenance of this kind of spatial resource exploitation. In the present study, by using Drosophila-parasitoid interactions as a model system, we tested the hypothesis whether intraspecific aggregation reflects an adaptive response to natural enemies. Most of the studies that have hitherto been carried out on Drosophila-parasitoid interactions used an almost two-dimensional artificial host environment, where host larvae could not escape from parasitoid attacks, and have demonstrated positive density-dependent parasitism risk. To test whether these studies captured the essence of such interactions, we used natural breeding substrates (decaying fruits). In a first step, we analysed the parasitism risk of Drosophila larvae on a three-dimensional substrate in natural fly communities in the field, and found that the risk of parasitism decreased with increasing host larval density (inverse density dependence). In a second step, we analysed the parasitism risk of Drosophila subobscura larvae on three breeding substrate types exposed to the larval parasitoids Asobara tabida and Leptopilina heterotoma. We found direct density-dependent parasitism on decaying sloes, inverse density dependence on plums, and a hump-shaped relationship between fly larval density and parasitism risk on crab apples. On crab apples and plums, fly larvae benefited from a density-dependent refuge against the parasitoids. While the proportion of larvae feeding within the fruit tissues increased with larval density, larvae within the fruit tissues were increasingly less likely to become victims of parasitoids than those exposed at the fruit surface. This suggests a facilitating effect of group-feeding larvae on reaching the spatial refuge. We conclude that spatial aggregation in Drosophila communities can at least in part be explained as a predator avoidance strategy, whereby natural enemies act as selective agents maintaining spatial patterns of resource utilisation in their host communities.     

Comparative Evaluation of the Gut Microbiota Associated with the Below- and Above-Ground Life Stages (Larvae and Beetles) of the Forest Cockchafer,Melolontha hippocastani

A comparison of the diversity of bacterial communities in the larval midgut and adult gut of the European forest cockchafer (Melolontha hippocastani) was carried out using approaches that were both dependent on and independent of cultivation. Clone libraries of the 16S rRNA gene revealed 150 operational taxonomic units (OTUs) that belong to 11 taxonomical classes and two other groups that could be classified only to the phylum level. The most abundant classes were β, δ and γ-proteobacteria, Clostridia, Bacilli, Erysipelotrichi and Sphingobacteria. Although the insect’s gut is emptied in the prepupal stage and the beetle undergoes a long diapause period, a subset of eight taxonomic classes from the aforementioned eleven were found to be common in the guts of diapausing adults and the larval midguts (L2, L3). Moreover, several bacterial phylotypes belonging to these common bacterial classes were found to be shared by the larval midgut and the adult gut. Despite this, the adult gut bacterial community represented a subset of that found in the larvae midgut. Consequently, the midgut of the larval instars contains a more diverse bacterial community compared to the adult gut. On the other hand, after the bacteria present in the larvae were cultivated, eight bacterial species were isolated. Moreover, we found evidence of the active role of some of the bacterial species isolated in food digestion, namely, the presence of amylase and xylanolytic properties. Finally, fluorescence in situ hybridization allowed us to confirm the presence of selected species in the insect gut and through this, their ecological niche as well as the metagenomic results. The results presented here elucidated the heterogeneity of aerobic and facultative bacteria in the gut of a holometabolous insect species having two different feeding habits.     

Leaf-Associated Bacterial and Fungal Taxa Shifts in Response to Larvae of the Tree Hole Mosquito, <Emphasis Type="Italic">Ochlerotatus triseriatus</Emphasis>

Larvae of the eastern tree hole mosquito, Ochlerotatus triseriatus (Say), and related container-breeding species are known to feed upon substrate-associated microorganisms. Although the importance of these microbial resources to larval growth has been established, almost nothing is known about the taxonomic composition and dynamics of these critical microbial food sources. We examined bacterial and fungal community compositional changes on oak leaves tethered in natural tree hole habitats of O. triseriatus. We eliminated larvae experimentally in a subset of the tree holes and examined 16S rDNA gene sequences for bacteria and ergosterol concentrations and 18S rRNA gene sequences for fungi collected from leaf material subsamples. Leaf ergosterol content varied significantly with time, but not treatment. Principal component analysis (PCA) was used to compare microbial taxonomic patterns found in leaves incubated with or without larvae present, and we found that larval presence affected both bacterial and fungal groups, either from loosely attached or strongly adherent categories. Bacterial communities generally grouped more tightly when larvae were present, and class level taxa proportions changed when larvae were present, suggesting selection by larval feeding or activities for particular taxa such as members of the Bacteroidetes, Alphaproteobacteria, and Betaproteobacteria classes. Fungal taxa composite scores also separated along PC axes related to the presence of larvae and indicated larval feeding effects on several higher taxonomic groups, including Saccharomycetes, Dothideomycetes, and Chytridiomycota. These results support the hypothesis that larval mosquito feeding and activities altered microbial communities associated with substrate surfaces, potentially leading to decreased food value of the resource and affecting decomposition of particulate matter in the system.     

Comparison of development of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus in mosquito larvae

Immunofluorescent staining was used with thin sections of paraffin-embedded specimens to detect the development of Bacillus thuringiensis var. israelensis and Bacillus sphaericus in the gut of mosquito larvae. The third- and fourth-instar larvae of Aedes aegypti, Anopheles maculatus, and Culex quinquefasciatus were fed either vegetative cells or spores of the bacteria. Spore germination, multiplication, and sporulation were studied in the larvae of each species. The spores of B. thuringiensis var. israelensis and B. sphaericus strain 2297 could germinate and cells could sporulate in the larval body. The vegetative cells of B. sphaericus strain 810428 were also able to produce spores in the mosquito larval gut, but the germination of spores could not be detected in the larvae. Multiplication of all bacterial species was observed after the larvae died. Growth of the bacteria in distilled water containing crude extracts of larvae made from each species was compared with that in synthetic medium (nutrient broth). They could produce spores and toxins in all the media used and the toxins had larvicidal activity against the target mosquitos Ae. aegypti, An. maculatus, and C. quinquefasciatus.     

Sensitivity to larval density in populations of Drosophila mojavensis: Influences of host plant variation on components of fitness

Summary Chromosomally polymorphic populations of Drosophila mojavensis from Baja California feed and breed on agria cactus, Stenocereus gummosus; whereas, monomorphic Arizona populations are associated exclusively with organ pipe cactus, S. thurberi. The effects of this host plant shift in expanding the kinds of feeding and breeding sites were assessed by manipulating larval density and recording differences in egg to adult development time and viability, and adult thorax size in both populations on artificially rotted substrates of both cactus species. Older agria rots increased development time but had no effect on viability. Organ pipe rots were qualitatively poorer substrates than agria rots for both monomorphic and polymorphic populations of D. mojavensis, especially at higher larval densities causing longer egg to adult development times, lower viabilities, and smaller thorax sizes than agria.The Baja population expressed shorter development times, higher viabilities, and smaller thorax sizes than the Arizona population on both cactus substrates. No evidence for cactus host race formation was found. The Baja population was less sensitive to increasing larval densities for all fitness characters studied on both cactus substrates indicating greater developmental homeostasis than in the monomorphic Arizona population. These data support the hypothesized central-marginal population structure within this species coincident with the distribution of host plants and lend insight into the process of adaptive divergence at different life history stages caused by host plant shifts.     

The culturable bacterial community of frass produced by larvae of Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) in the Canary island date palm

Aims: Larvae of the red palm weevil (RPW) Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) feed inside palm stem tissues, making galleries and producing a wet fermenting frass. We characterized the culturable bacteria associated with frass produced by tunnelling larvae inside the Canary island date palms and investigated the role of frass and gut bacteria in plant polymers breakdown. Methods and Results: A culture‐dependent method was used to isolate bacteria from frass and noninfested palm tissues. Bacterial isolates were grouped into operational taxonomic units based on polymorphisms in the ITS‐PCR profiles, and representative isolates were identified by partial sequencing of the 16S rRNA gene. Frass bacteria were dominated by 2,3‐butanediol fermenter Enterobacteriaceae. None of the bacterial isolates was able to degrade cellulose; however, cellulolytic and hemicellulolytic bacteria were isolated from the larval gut enrichment cultures. Conclusions: Frass bacteria are specifically associated with the RPW larvae and might play beneficial roles for RPW, other than nutritional, that deserve further investigations. Breakdown of plant polymers probably occurs inside the larvae digestive system. Significance and Impact of the Study: Frass and gut micro‐organisms of R. ferrugineus should be included in studies of the interactions between RPW, its plant hosts, and its enemies.     

Allometric growth and functional development of the gut in developing cod Gadus morhua L. larvae

The development of the gut epithelium in cod Gadus morhua was studied during the larval period in intensive rearing systems. Cod larvae were fed enriched rotifers from mouth opening. On 17 days post‐hatch (dph) one group of larvae were fed Artemia sp. nauplii while another group were fed both rotifers and a formulated diet (co‐fed). At the end of the experiment (30 dph) larvae receiving live feed were almost three times larger than the co‐fed larvae, although no clear signs of pathological effects due to feeding regime were found in any larvae sampled for morphological studies. The midgut volume in larvae fed live feed increased by a factor of 38 during the experiment, and in particular volume increased rapidly between 24 and 30 dph. The enterocyte size increased between 12 and 24 dph from 652 ± 64 to 1479 ± 144 μm³ (mean ±s .e .). When enterocytes reached their maximum size, several morphological changes in the gut epithelium were initiated, such as increased number of mitochondria per enterocyte, increased size of the nuclei and a considerable increase in microvilli surface area. The mitochondrial membrane structures changed during the experiment, suggesting a maturation process of the mitochondria. The midgut development was strongly related to larval size rather than age. On 30 dph co‐fed larvae were equal in size to Artemia sp. fed larvae on 24 dph. This was reflected by equal values of estimated midgut volume, midgut length and total number of enterocytes and the number of mitochondria per enterocyte. The microvilli surface area, however, was significantly larger in co‐fed larvae on 24 dph compared to live‐feed larvae on 30 dph. This increase in absorptive surface was probably a response to suboptimal feeding conditions. The strong correlation between gut development and larval size and the lack of clear pathological effects, suggested that the gut tissue is flexible and can withstand periods of suboptimal nutrition at this stage.     

Microbial Gut Diversity of Africanized and European Honey Bee Larval Instars

The first step in understanding gut microbial ecology is determining the presence and potential niche breadth of associated microbes. While the core gut bacteria of adult honey bees is becoming increasingly apparent, there is very little and inconsistent information concerning symbiotic bacterial communities in honey bee larvae. The larval gut is the target of highly pathogenic bacteria and fungi, highlighting the need to understand interactions between typical larval gut flora, nutrition and disease progression. Here we show that the larval gut is colonized by a handful of bacterial groups previously described from guts of adult honey bees or other pollinators. First and second larval instars contained almost exclusively Alpha 2.2, a core Acetobacteraceae, while later instars were dominated by one of two very different Lactobacillus spp., depending on the sampled site. Royal jelly inhibition assays revealed that of seven bacteria occurring in larvae, only one Neisseriaceae and one Lactobacillus sp. were inhibited. We found both core and environmentally vectored bacteria with putatively beneficial functions. Our results suggest that early inoculation by Acetobacteraceae may be important for microbial succession in larvae. This assay is a starting point for more sophisticated in vitro models of nutrition and disease resistance in honey bee larvae.     

The nonfeeding auricularia of Holothuria mexicana (Echinodermata,Holothuroidea)

Among echinoderms, nonfeeding larvae usually are simplified in body shape, have uniform ciliation, and have lost the larval gut. A few species have nonfeeding larvae that express some remnant features of feeding larvae like ciliated bands and larval skeleton or larval arms, but typically their larval mouth never opens and their gut does not function. Still other species have retained the feeding larval form, a functional gut, and can feed, but they do not require food to metamorphose. The present note describes the development of a tropical holothurian, Holothuria mexicana, which hatches as a gastrula that is already generating coelomic structures. A translucent auricularia forms with a mouth that opens but becomes reduced soon thereafter. In form and ciliation this auricularia resembles a feeding larva, but it does not respond to food. A doliolaria forms on day 4 and the pentactula on day 6 post‐fertilization. Further study of this larva and that of its closely related congener, Holothuria floridana, is warranted.     

Bacterial Infection and Immune Responses in Lutzomyia longipalpis Sand Fly Larvae Midgut

The midgut microbial community in insect vectors of disease is crucial for an effective immune response against infection with various human and animal pathogens. Depending on the aspects of their development, insects can acquire microbes present in soil, water, and plants. Sand flies are major vectors of leishmaniasis, and shown to harbor a wide variety of Gram-negative and Gram-positive bacteria. Sand fly larval stages acquire microorganisms from the soil, and the abundance and distribution of these microorganisms may vary depending on the sand fly species or the breeding site. Here, we assess the distribution of two bacteria commonly found within the gut of sand flies, Pantoea agglomerans and Bacillus subtilis. We demonstrate that these bacteria are able to differentially infect the larval digestive tract, and regulate the immune response in sand fly larvae. Moreover, bacterial distribution, and likely the ability to colonize the gut, is driven, at least in part, by a gradient of pH present in the gut.     

Comparison of the gut bacterial flora of start-feeding larval turbot reared under different conditions

P.D. MUNRO, A. BARBOUR AND T.H. BIRKBECK. 1994. Bacterial colonization of the gut of turbot larvae coincided with the start of feeding and the gut microflora was then dominated by Vibrio and Aeromonas species. The detection of similar bacterial isolates over several days indicated the presence of a stable microflora during the rotifer feeding stage, probably reflecting a stable flora in the rotifer culture. There was no correlation between the number of bacteria in the gut and larval survival rates; incidences of high mortalities were not associated with high numbers of recognized fish pathogens, although an Aeromonas species was implicated as an opportunistic pathogen. Potential pathogens were isolated, albeit in low numbers, from batches of apparently healthy larvae with high survival rates. It is probable that the bacterial flora plays an important role in determining the survival of larval fish and that the establishment of a beneficial flora is possible, although the criteria for such a flora have still to be elucidated.     

Effect of ace inhibitors and TMOF on growth, development, and trypsin activity of larval Spodoptera littoralis

Angiotensin converting enzyme (ACE) is a zinc metallopeptidase capable of cleaving dipeptide or dipeptideamide moieties at the C-terminal end of peptides. ACE is present in the hemolymph and reproductive tissues of insects. The presence of ACE in the hemolymph and its broad substrate specificity suggests an important role in processing of bioactive peptides. This study reports the effects of ACE inhibitors on larval growth in the cotton leafworm Spodoptera littoralis. Feeding ACE inhibitors ad lib decreased the growth rate, inhibited ACE activity in the larval hemolymph, and down-regulated trypsin activity in the larval gut. These results indicate that S. littoralis ACE may influence trypsin biosynthesis in the larval gut by interacting with a trypsin-modulating oostatic factor (TMOF). Injecting third instar larvae with a combination of Aea-TMOF and the ACE inhibitor captopril, down-regulated trypsin biosynthesis in the larval gut indicating that an Aea-TMOF gut receptor analogue could be present. Injecting captopril and enalapril into newly molted fifth instar larvae stopped larval feeding and decreased weight gain. Together, these results indicate that ACE inhibitors are efficacious in stunting larval growth and ACE plays an important role in larval growth and development.     

Identification and ecology of bacterial communities associated with necroses of three cactus species.

To compare the bacterial communities residing in necrotic tissues of columnar cacti of the Sonoran Desert, isolates from 39 organ pipe, 19 saguaro, and 16 senita cacti were obtained. The isolates were clustered into 28 conspecific groups on the basis of their fatty acid profiles. The distributions of the individual bacterial isolates varied among cactus species. Seven of the 28 species groups were unique to a particular cactus species, whereas 8 species groups were found in all three cacti. The effective number of bacterial species for each cactus species was positively correlated with both the chemical complexity and glucose concentration of the plant tissues. The effective number of bacterial species and bacterial distribution patterns were compared with those known for communities of cactophilic yeasts. The observed bacterial distribution patterns are most likely due to differences in the chemical compositions of the three cactus species.