Yeasts Isolated from Neotropical Wood-Boring Beetles in SE Peru

Some temperate wood-boring cerambycid beetles harbor intracellular gut yeasts believed to augment host nutrition, but species belonging to the subfamily Lamiinae are thought to lack endosymbionts. Almost 49 percent of Neotropical cerambycid species are lamiines, therefore, comparatively few rain forest species would be expected to host symbiotic gut yeasts. This study reports the isolation of gut yeasts from closely related Neotropical lamiines. We investigated species that feed on trees in the Brazil nut family (Lecythidaceae), because host plant associations are relatively well known. Our objectives were to determine if gut yeasts were present and, if possible, infer their mode of transmission. We collected and dissected 18 beetle specimens from three tree species, including 17 cerambycids and one curculionid. Every insect specimen yielded a gut yeast. DNA sequence libraries were used for a rapid identification of the yeasts and their larval hosts. The cerambycids included five lamiine species and one cerambycine. Six ascomycete yeasts were isolated from their guts; we found no evidence of strict vertical transmission. Larval gut yeasts were genetically similar to yeasts previously isolated from insects associated with wood or fungi, implying potential habitat specificity. The yeasts have not yet been localized, and potential function is not known, but they may contribute to rapid nutrient cycling or serve as the first line of defense against plant toxins.     

Rearing of the colydiid beetle predator, Dastarcus helophoroides, on artificial diet

Artificial diets for the colydiid beetle, Dastarcus helophoroides, a predator of cerambycid beetles and xylocopid bees, were evaluated. Hatched larvae were reared on artificial diets composed of silkworm pupa-powder, dry yeasts, yeast extract, sucrose, peptone, squid liver oil, preservatives and distilled water, but their emergence rate was very low. If larvae were fed paralyzed cerambycid larvae till they became approximately 8 mm in body length and then reared on artificial diet, emergence rates were high. Using the latter method, mass-production of this colydiid beetle is feasible.     

Developmental expression of Manduca sexta hemolin.

Hemolin is hemolymph protein that is a member of the immunoglobulin superfamily. Its induced expression after bacterial infection suggests that it functions in the immune response. In this paper, we describe the expression of the Manduca sexta hemolin gene at certain developmental stages in the absence of microbial challenge. Hemolin was present at a very low level in hemolymph of naive larvae until the beginning of the wandering stage prior to pupation, when its concentration in hemolymph increased dramatically. At the same time, hemolin could be found in the fluid contained in the midgut lumen. The appearance of hemolin mRNA in fat body and midgut at the beginning of the wandering stage correlated with the presence of hemolin in the hemolymph and midgut lumen. Hemolin was present in hemolymph through the pupal and adult stages. Hemolin was also present in newly deposited eggs, and persisted in eggs throughout embryonic development. A hemolin cDNA isolated from an adult fat body library had the same sequence as those previously obtained from larval libraries. Hemolin purified from hemolymph of bacteria-injected larvae, from hemolymph of naive wandering stage larvae and adult moths, and from midgut fluid of wandering stage larvae had the same apparent mass, which was consistent with the mass predicted from the hemolin cDNA sequence. Hemolin from hemolymph of wandering stage larvae did not contain any detectable carbohydrate, but hemolin from the hemolymph of bacteria-injected larvae and from naive adult moths was associated with carbohydrate, although of different amounts and composition. These results suggest that a single hemolin gene is developmentally regulated and is also induced when insects are exposed to microbial infection. M. sexta hemolin apparently lacks post-translational covalent glycosylation, but instead is associated under some conditions with non-covalently bound carbohydrates. Arch.     

Activated host neutrophils in the larval midgut lumen of the human bot fly Dermatobia hominis

Light microscopy (LM) and transmission electron microscopy (TEM) were used to observe activated polymorphonuclear neutrophils from mammalian hosts as well as invading bacteria in the midgut lumen of larvae of the human bot fly Dermatobia hominis. Other resident or recruited cells associated with dermal myiasis were fed on by larvae and digested more rapidly than neutrophils. The latter were observed moving towards bacteria and particles of food, extending the filopodia and engulfing material to be digested within phagosomes. The larval midgut lumen, thus, appears to be a suitable environment to produce neutrophil activation at least for short periods, as seen in mammalian hosts. Although interactions between phagocytes and bacteria in the midgut lumen may be important in bot fly larval development, further studies are required to confirm this.     

Cloning and expression analysis of midgut chymotrypsin-like proteinases in the tobacco hornworm

Digestion of proteins in the midgut of lepidopteran larvae relies on different trypsin and chymotrypsin isoforms. In this study we describe three chymotrypsin-like proteinases (CTLP2-4) from the larval midgut of Manduca sexta, which are closely related to CTLP1 and less closely related to another chymotrypsin (CT), two previously described proteinases present in the larval midgut of M. sexta. CTLP1-4 fit perfectly into a novel subgroup of insect CTLPs by sequence similarity and by the replacement of GP by SA in the highly conserved GDSGGP motif. When we examined CTLP expression in different tissues, most of the proteinases were predominantly expressed in the anterior and median midgut, while some were found in the Malpighian tubules. When we examined CTLP expression at different physiological states, we observed that the CTLP mRNA amounts did not differ considerably in feeding and starving larvae except for CTLP2, whose mRNA dropped significantly upon starvation. During moulting, however, the mRNA amounts of all CTLPs dropped significantly. When we immunologically examined CTLP amounts, mature proteinases were only detectable in the gut lumen of feeding and re-fed larvae, but not in that of starving or moulting larvae, suggesting that CTLP secretion is suspended during starvation or moult.     

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.     

Potential role for gut microbiota in cell wall digestion and glucoside detoxification in Tenebrio molitor larvae

Tenebrio molitor larvae were successfully reared free of cultivatable gut lumen bacteria, yeasts and fungi using two approaches; aseptic rearing from surface sterilized eggs and by feeding larvae with antibiotic-containing food. Insects were reared on a rich-nutrient complete diet or a nutrient-poor refractory diet. A comparison of digestive enzyme activities in germ free and conventional insects containing a gut microbiota did not reveal gross differences in enzymes that degrade cell walls from bacteria (lysozyme), fungi (chitinase and laminarinase) and plants (cellulase and licheninase). This suggested that microbial-derived enzymes are not an essential component of the digestive process in this insect. However, more detailed analysis of T. molitor midgut proteins using an electrophoretic separation approach showed that some digestive enzymes were absent and others were newly expressed in microbiota-free larvae. Larvae reared in antibiotic-containing refractory wheat bran diet performed poorly in comparison with controls. The addition of saligenin, the aglycone of the plant glucoside salicin, has more deleterious effects on microbiota-free larvae than on the conventionally reared larvae, suggesting a detoxifying role of midgut microbiota. Analysis of the volatile organic compounds released from the faecal pellets of the larvae shows key differences in the profiles from conventionally reared and aseptically reared larvae. Pentadecene is a semiochemical commonly found in other beetle species. Here we demonstrate the absence of pentadecene from aseptically reared larvae in contrast to its presence in conventionally reared larvae. The results are discussed in the light of the hypothesis that microbial products play subtle roles in the life of the insect, they are involved in the digestion of refractory food, detoxification of secondary plant compounds and modify the volatile profiles of the insect host.     

Always being well prepared for defense: The production of deterrents by juvenile Chrysomelina beetles (Chrysomelidae)

In response to herbivores, plants produce a variety of natural compounds. Many beetle species have developed ingenious strategies to cope with these substances, including colonizing habitats not attractive for other organisms. Leaf beetle larvae of the subtribe Chrysomelina, for example, sequester plant-derived compounds and use them for their own defense against predators. Using systematically modified structural mimics of plant-derived glucosides, we demonstrated that all tested Chrysomelina larvae channel compounds from the gut lumen into the defensive glands, where they serve as intermediates in the synthesis of deterrents. Detailed studies of the sequestration process revealed a functional network of transport processes guiding phytochemicals through the larval body. The initial uptake by the larvae’s intestine seems to be fairly unspecific, which contrasts sharply with the specific import of precursors into the defensive glands. The Malpighian tubules and hind-gut organs facilitate the rapid clearing of body fluid from excess or unusable compounds. The network exists in both sequestering species and species producing deterrents de novo. Transport proteins are also required for de novo synthesis to channel intermediates from the fat body to the defensive glands for further conversion. Thus, all the tools needed to exploit host plants’ chemistry by more derived Chrysomelina species are already developed by iridoid–de novo producers. Early intermediates from the iridoid–de novo synthesis which also can be sequestered are able to regulate the enzyme activity in the iridoid metabolism.     

Effect of different holding regimens on the intestinal microflora of herring (Clupea harengus) larvae.

The aerobic intestinal microflora of 2-week-old herring (Clupea harengus) larvae was characterized by using conventional microbiological methods and electron microscopy. Larvae were hatched and kept in filtered seawater or in seawater with penicillin and streptomycin. The gastrointestinal tract of herring larvae is essentially a straight tube divided into two compartments. Light microscopy revealed bacteria present in a progressively increasing amount throughout the length of the gastrointestinal tract from esophagus to anus. The posterior region of the intestinal lumen appeared completely occluded with bacteria. The intestinal microflora consisted mainly of members of the genera Pseudomonas and Alteromonas in the larvae incubated in filtered seawater, whereas Flavobacterium spp. dominated in larvae exposed to antibiotics. The intestinal microflora of untreated fish larvae was sensitive to all tested antibiotics, whereas multiple resistance was found in the intestinal microflora of the group given antibiotics. Thus, a dramatic change in the microflora resulted from incubation with antibiotics. Nonpigmented yeasts were detected in both larval groups. Ciliated epithelial cells were observed in the midgut, probably propeling bacteria towards the hindgut, where endocytosis of bacteria has been demonstrated. These findings suggest that transport and sequestering mechanisms resembling those of invertebrates may be found in the gut of fish larvae. The possible significance for larval health and nutrition is discussed.     

Active Na+ uptake in the isolated midgut of larval Sarcophaga bullata

The isolated midgut of larval Sarcophaga bullata actively accumulates Na⁺ from the gut lumen into the haemolymph. The active transport of Na⁺ out of the gut lumen is responsible for the transepithelial potential difference measured across the midgut epithelium, such that the midgut lumen is negative in respect to the haemolymph side. Both the net movement of Na⁺ out of the midgut lumen and the transepithelial potential are inhibited by CN^? and, in addition, the potential in blocked by ouabain.     

Effect of different holding regimens on the intestinal microflora of herring (Clupea harengus) larvae.

The aerobic intestinal microflora of 2-week-old herring (Clupea harengus) larvae was characterized by using conventional microbiological methods and electron microscopy. Larvae were hatched and kept in filtered seawater or in seawater with penicillin and streptomycin. The gastrointestinal tract of herring larvae is essentially a straight tube divided into two compartments. Light microscopy revealed bacteria present in a progressively increasing amount throughout the length of the gastrointestinal tract from esophagus to anus. The posterior region of the intestinal lumen appeared completely occluded with bacteria. The intestinal microflora consisted mainly of members of the genera Pseudomonas and Alteromonas in the larvae incubated in filtered seawater, whereas Flavobacterium spp. dominated in larvae exposed to antibiotics. The intestinal microflora of untreated fish larvae was sensitive to all tested antibiotics, whereas multiple resistance was found in the intestinal microflora of the group given antibiotics. Thus, a dramatic change in the microflora resulted from incubation with antibiotics. Nonpigmented yeasts were detected in both larval groups. Ciliated epithelial cells were observed in the midgut, probably propeling bacteria towards the hindgut, where endocytosis of bacteria has been demonstrated. These findings suggest that transport and sequestering mechanisms resembling those of invertebrates may be found in the gut of fish larvae. The possible significance for larval health and nutrition is discussed.     

嗜菌异小杆线虫侵染后暗黑鳃金龟和大黑鳃金龟幼虫脂肪体和中肠组织超微结构观察

【目的】探究昆虫病原线虫嗜菌异小杆线虫沧州品系 Heterorhabditis bacteriophora strain Cangzhou侵染对蛴螬脂肪体和中肠的影响,进一步明确其对蛴螬的致病机理。【方法】采用透射电镜技术,观察暗黑鳃金龟 Hololtrichia oblita (Faldermann)和大黑鳃金龟 H. parallela Motschulsky 2龄幼虫被嗜菌异小杆线虫沧州品系侵染后其脂肪体和中肠组织的病理变化。【结果】血腔注射感染期病原线虫嗜菌异小杆线虫沧州品系悬浮液24和48 h后,观察发现暗黑鳃金龟和大黑鳃金龟2龄幼虫脂肪体和中肠的组织结构均按时序逐渐发生变化,起初表现为脂肪球变形或变小,颜色变浅,脂肪体细胞和中肠细胞内质网、线粒体肿胀,中肠微绒毛变形脱落等现象,48 h后包裹脂肪球的膜结构破裂,脂肪体细胞和中肠细胞线粒体破裂,内质网数量减少,中肠微绒毛大量脱落,同时核内染色质大量解离,核膜破裂。【结论】经昆虫病原线虫嗜菌异小杆线虫沧州品系处理后,暗黑鳃金龟和大黑鳃金龟两种金龟甲2龄幼虫脂肪体和中肠细胞均出现明显的病理变化过程,这是嗜菌异小杆线虫高效致死蛴螬的原因之一。本研究可为昆虫病原线虫作为一种生物防治手段在蛴螬的综合防治中更好地发挥作用提供理论依据。     

Lysozyme in the midgut of Manduca sexta during metamorphosis.

Low levels of lysozyme were found in the midgut epithelium of the tobacco hornworm, Manduca sexta, during the early part of the fifth larval stadium. This was observed in control insects as well as in bacterially challenged insects. No lysozyme was detected in the gut contents of either group of insects which were actively eating or in the early stages of metamorphosis. However, high levels of lysozyme activity were detected in homogenates of midgut tissue collected from insects later in the stadium. Immunocytochemical studies demonstrated that lysozyme accumulates in large apical vacuoles in regenerative cells of the midgut during the larval-pupal molt. These cells, initially scattered basally throughout the larval midgut epithelium, multiply and form a continuous cell layer underneath the larval midgut cells. At the larval/pupal ecdysis the larval midgut epithelium is sloughed off and the regenerative cells, now forming the single cell layer of the midgut, release the contents of their vacuoles into the midgut lumen. This release results in high lysozyme activity in the lumen of the pupal midgut and is thought to confer protection from bacterial infection. This is the first indication that the lysozyme gene may be developmentally regulated in a specific tissue in the absence of a bacterial infection.     

In vitro and in vivo binding of snowdrop (Galanthus nivalis agglutinin; GNA) and jackbean (Canavalia ensiformis; Con A) lectins within tomato moth (Lacanobia oleracea) larvae; mechanisms of insecticidal action

When fed in semi-artificial diet the lectins from snowdrop (Galanthus nivalis: GNA: mannose-specific) and jackbean (Canavalia ensiformis: Con A: specific for glucose and mannose) were shown to accumulate in vivo in the guts, malpighian tubules and haemolymph of Lacanobia oleracea (tomato moth) larvae. Con A, but not GNA, also accumulated in the fat bodies of lectin-fed larvae. The presence of glycoproteins which bind to both lectins in vitro was confirmed using labelled lectins to probe blots of polypeptides extracted from larval tissues. Immunolocalisation studies revealed a similar pattern of GNA and Con A binding along the digestive tract with binding concentrated in midgut sections. Binding of lectins to microvilli appeared to lead to transport of the proteins into cells of the gut and malpighian tubules. These results suggested that both lectins are able to exert systemic effects via transport from the gut contents to the haemolymph across the gut epithelium. The delivery of GNA and Con A to the haemolymph was shown to be dependent on their functional integrity by feeding larvae diets containing denatured lectins. Con A, but not GNA, was shown to persist in gut and fat body tissue of lectin-fed larvae chased with control diet for three days. Con A also shows more extensive binding to larval tissues in vitro than GNA, and these two factors are suggested to contribute to the higher levels of toxicity shown by Con A, relative to GNA, in previous long term bioassays.     

Stage-specific distribution of oxidative radicals and antioxidant enzymes in the midgut of Leptinotarsa decemlineata

The titers of reactive oxygen species (ROS) represented by superoxide anion and general peroxides, and the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), are regulated in the midgut of the Colorado potato beetle (CPB) relative to the gut compartment, developmental stage, and food intake. ROS concentration is low in the potato leaves but it is very high in their digest in insect's anterior midgut. It is proposed that intensive ROS production in this gut region is linked to the processing of allelochemicals. SOD and CAT activities, low oxygen tension, and unidentified redox systems that maintain a slightly reducing milieu in the midgut lumen (pe+pH=6.95 declining to 5.36), obviously contribute to the decrease of ROS concentration along the gut length to a minimum in the wall of posterior midgut region. SOD and CAT activities are higher in the potato leaves than in the midgut tissues but the role of plant enzymes in ROS elimination within the gut lumen remains to be shown. A lower level of ROS and a higher antioxidant potential in the adult than in the larval midgut indicate stage specificity in the management of oxidative stress. The antioxidant defense is high in the diapausing adults that contain no detectable superoxide and about ten times less peroxides than the reproducing adults.     

Transepithelial flux of an allatostatin and analogs across the anterior midgut of Manduca sexta larvae in vitro

The transepithelial flux of cydiastatin 4 and analogs across flat sheet preparations of the anterior midgut of larvae of the tobacco hawkmoth moth, Manduca sexta, was investigated using a combination of reversed-phase high-performance liquid chromatography (RP-HPLC), enzyme-linked immunosorbent assay (ELISA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The lumen to hemolymph (L-H) flux of cydiastatin 4 was dose and time-dependent, with a maximum rate of flux of c. 178 pmol/cm2/h) measured after a 60-min incubation with 100 micromol/l of peptide in the lumen bathing fluid. The rates of flux, L-H and H-L, across the isolated gut preparations were not significantly different. These data suggest that uptake across the anterior midgut of larval M. sexta is via a paracellular route. Cydiastatin 4 was modified to incorporate a hexanoic acid (Hex) moiety at the N-terminus, the N-terminus extended with 5 P residues and/or the substitution of G7 with Fmoc-1-amino-cyclopropylcarboxylic acid (Acpc). The incorporation of hexanoic acid enhanced the uptake of these amphiphilic analogs compared to the native peptide. Analogs were also more resistant to enzymes in hemolymph and gut preparations from larval M. sexta. A modified N-terminus gave protection against aminopeptidase-like activity and incorporation of Acpc inhibited endopeptidase-like activity. Although analogs were stable in the hemolymph, they were susceptible to amidase-like activity in the gut, which appears to convert the C-terminal amide group to a free carboxylic acid, identified by an increase in 1 mass unit of the peptide analog.     

A larval serum protein of the silkworm,Bombyx mori: cDNA sequence and developmental specificity of the transcript

The Bombyx mori larval serum protein (BmLSP) is a major component of larval hemolymph proteins until early in the last instar. The cDNA for BmLSP was cloned from a library constructed from fat body RNA of penultimate instar larvae, and the complete nucleotide sequence of the 909 base pair cDNA insert was determined. The deduced 262 amino acid polypeptide included a 16 amino acid residue signal peptide and a 15 amino acid sequence prosegment. A homology search showed that BmLSP has significant similarity with microvitellogenin of Manduca sexta and the 30K proteins of B. mori. Tissue distribution and developmental profile of BmLSP mRNA were analyzed by northern hybridization. BmLSP mRNA was abundant in fat body but not detected in midgut and silk gland. BmLSP mRNA was present during the feeding periods of the fourth and fifth instar larvae, but absent during the larval molt and after the onset of cocoon spinning.     

The yeast Wickerhamomyces anomalus (Pichia anomala) inhabits the midgut and reproductive system of the Asian malaria vector Anopheles stephensi

While symbiosis between bacteria and insects has been thoroughly investigated in the last two decades, investments on the study of yeasts associated with insects have been limited. Insect-associated yeasts are placed on different branches of the phylogenetic tree of fungi, indicating that these associations evolved independently on several occasions. Isolation of yeasts is frequently reported from insect habitats, and in some cases yeasts have been detected in the insect gut and in other organs/tissues. Here we show that the yeast Wickerhamomyces anomalus, previously known as Pichia anomala, is stably associated with the mosquito Anopheles stephensi, a main vector of malaria in Asia. Wickerhamomyces anomalus colonized pre-adult stages (larvae L(1)-L(4) and pupae) and adults of different sex and age and could be isolated in pure culture. By a combination of transmission electron microscopy and fluorescent in situ hybridization techniques, W. anomalus was shown to localize in the midgut and in both the male and female reproductive systems, suggesting multiple transmission patterns.     

Microbial associations in gut systems of wood- and bark-inhabiting longhorned beetles [Coleoptera: Cerambycidae]

Using fluorescence in situ hybridization (FISH) techniques and PCR-based rDNA sequencing, gut microflora in the larvae of bark- and wood-inhabiting cerambycid beetles (Rhagium inquisitor, Tetropium castaneum, Plagionotus arcuatus and Leptura rubra [Coleoptera: Cerambycidae]) was investigated. A total of 12 novel ascomycetous yeast strains were isolated from the gut content. Panfungal and strain-specific oligonucleotide probes identified two yeast strains as Candida rhagii and Candida shehatae, which were colonizing specialized organs (mycetomes) adhering to the gut of R. inquisitor and L. rubra larvae, respectively. Fragments containing these organisms were constantly being released from the mycetomes into the gut lumen. Whereas the mycetome symbiont of T. castaneum could not be identified, all larvae of this species harbored an additional bacterial endocytobiont in their gut epithelium. This novel gammaproteobacterium belonged to the Sodalis clade of insect symbionts, which includes the secondary endosymbiont of tsetse flies (Sodalis glossinidius) and the Sitophilus oryzae primary endosymbiont (SOPE).