Beetle Gut Research Articles

Contrasts in Cellulolytic Activities of Gut Microorganisms Between the Wood Borer,Saperda vestita(Coleoptera: Cerambycidae), and the Bark Beetles,Ips piniandDendroctonus frontalis(Coleoptera: Curculionidae)

The goal of this study was to characterize the cellulose-degrading microorganisms in the guts of wood-inhabiting beetles. We enriched for cellulose-degrading microorganisms by inoc- ulating Þlter paper in liquid growth medium with macerated guts from larvae and adults of the wood-boring longhorned beetle, Saperda vestita, and the phloeophagous bark beetles, Ips pini and Dendroctonus frontalis. After incubation, microorganisms were isolated in pure culture and tested for their ability to digest carboxymethylcellulose (CMC). Isolates were identiÞed based on their ribo- somal RNA gene or intergenic region sequences. Bacteria and fungi from the gut ofS. vestita degraded Þlter paper, whereas the microorganisms from the phloeophagous insects did not. The only bacterium detected that was able to degrade CMC is closely related to Sphingobium yanoikuyae, a member of the-Proteobacteria class. We found this species in all Þeld-collectedS.vestita larvae in 2002 and 2003. Population densities of cellulolytic bacteria in S. vestita ranged from 2.4 10 5 to 3.6 10 6 CFU/gut. Bacteria isolated from the phloeophagous beetles I. pini and D. frontalis did not degrade CMC. Two fungi isolated from the gut of S. vestita adults had strong degradative activity. Sequences of the ITS1, 5.8S, and ITS2 rRNA regions indicated that these fungi are highly similar to Fusarium culmorum and Penicillium crustosum, respectively. This study provides the Þrst description of the gut microbial community ofS.vestita and the Þrst documentation of association between cellulolytic microorganisms and a wood-boring beetle.

The beetle gut: a hyperdiverse source of novel yeasts

We isolated over 650 yeasts over a three year period from the gut of a variety of beetles and characterized them on the basis of LSU rDNA sequences and morphological and metabolic traits. Of these, at least 200 were undescribed taxa, a number equivalent to almost 30% of all currently recognized yeast species. A Bayesian analysis of species discovery rates predicts further sampling of previously sampled habitats could easily produce another 100 species. The sampled habitat is, thereby, estimated to contain well over half as many more species as are currently known worldwide. The beetle gut yeasts occur in 45 independent lineages scattered across the yeast phylogenetic tree, often in clusters. The distribution suggests that the some of the yeasts diversified by a process of horizontal transmission in the habitats and subsequent specialization in association with insect hosts. Evidence of specialization comes from consistent associations over time and broad geographical ranges of certain yeast and beetle species. The discovery of high yeast diversity in a previously unexplored habitat is a first step toward investigating the basis of the interactions and their impact in relation to ecology and evolution.

The status and characterization of Enteroramus dimorphus: a xylose-fermenting yeast attached to the gut of beetles

Enteroramus dimorphus from the gut of the passalid beetle Odontotaenius disjunctus was described originally as a yeast-like fungus of unknown taxonomic affiliation. The fungus can be observed in situ, attached by a specialized cell to the beetle hindgut wall. In a recent study of yeast endosymbionts from a variety of beetles, we discovered that E. dimorphus is a member of the Pichia stipitis (Saccharomycetes) clade, known for xylose fermentation and assimilation. The closest relative of E. dimorphus is the PASS1 isolate, repeatedly acquired from passalid beetles in eastern North America from Pennsylvania to Louisiana. In addition to xylose fermentation and assimilation, these yeasts are characterized by the production of hat-shaped ascospores in culture, assimilation of a wide range of sugars, and synthesis of several vitamins. Enteroramus dimorphus, however, can be distinguished from close relatives by several physiological characteristics and rDNA sequences, which vary slightly from the more widespread PASS1 genotype. We present an amended description of E. dimorphus and discuss its symbiotic phase in association with O. disjunctus, including a holdfast that parallels those of unrelated symbiotic yeasts associated with nematodes.

The Status and Characterization of Enteroramus dimorphus: A Xylose-Fermenting Yeast Attached to the Gut of Beetles

Enteroramus dimorphus from the gut of the passalid beetle Odontotaenius disjunctus was described originally as a yeast-like fungus of unknown taxonomic affiliation. The fungus can be observed in situ, attached by a specialized cell to the beetle hindgut wall. In a recent study of yeast endosymbionts from a variety of beetles, we discovered that E. dimorphus is a member of the Pichia stipitis (Saccharomycetes) clade, known for xylose fermentation and assimilation. The closest relative of E. dimorphus is the PASS1 isolate, repeatedly acquired from passalid beetles in eastern North America from Pennsylvania to Louisiana. In addition to xylose fermentation and assimilation, these yeasts are characterized by the production of hat-shaped ascospores in culture, assimilation of a wide range of sugars, and synthesis of several vitamins. Enteroramus dimorphus, however, can be distinguished from close relatives by several physiological characteristics and rDNA sequences, which vary slightly from the more widespread PASS1 genotype. We present an amended description of E. dimorphus and discuss its symbiotic phase in association with O. disjunctus, including a holdfast that parallels those of unrelated symbiotic yeasts associated with nematodes.

Microorganisms in the gut of beetles: evidence from molecular cloning

We have regularly cultured yeasts from the gut of certain beetles in our ongoing research. In this study cloned PCR products amplified from the gut contents of certain mushroom-feeding and wood-ingesting beetles in four families (Erotylidae, Tenebrionidae, Ciidae, and Passalidae) were sequenced and compared with culture results. Cultural techniques detected some yeasts present in the gut of the beetles, including a Pichia stipitis-like yeast associated with wood-ingesting passalid beetles. Clone sequences similar to several ascomycete yeasts and Malassezia restricta, a fastidious basidiomycetous yeast requiring special growth media, however, were not detected by culturing. Unexpectedly, phylogenetic analysis of additional clone sequences discovered from passalid beetles showed similarity to members of the Parabasalia, protists known from other wood-ingesting insects, termites, and wood roaches. Examination of all gut regions of living passalids, however, failed to reveal parabasalids, and it is possible that they were parasites in the gut tissue present in low numbers.

Field Persistence of Ice-Nucleating Bacteria in Overwintering Colorado Potato Beetles

Abstract Ice-nucleating bacteria are biological ice nucleators capable of elevating the temperature at which ice crystals form in the body fluids of insects, termed the supercooling point (SCP). In the freeze-intolerant Colorado potato beetle, these bacteria reduce its cold tolerance and, consequently, increase the likelihood of mortality in overwintering adults exposed to subzero temperatures. In this field study, two ice-nucleating bacteria, Pseudomonas fluorescens and P. putida, were evaluated for their persistence and efficacy against overwintering adults. Both strains significantly elevated the SCP of treated beetles immediately after ingestion. However, only beetles fed P. fluorescens still had significantly elevated SCPs (−4.2°C) versus control (−6.4°C) after overwintering in the field. Bacterial persistence in beetle guts was confirmed by PCR assays that positively correlated the presence of P. fluorescens with elevated SCPs. Despite the reduction of cold tolerance in overwintering adults with P. fluorescens, no significant difference was observed in the survival rates of treated versus control beetles during the winter. Because adults overwinter in the ground, the effect of this bacterium on beetle survival depends on the soil temperatures that overwintering adults experience. Nevertheless, recovery of most of the adults in the upper 15 cm of soil strata indicates that beetles with potent ice-nucleating bacteria in their guts could be subject to critically low temperatures during winter. Our results show that ice-nucleating P. fluorescens compromises the cold tolerance of Colorado potato beetles and suggest that by development of strategies to maximize bacterial efficacy in the field, this bacterium offers a potential biocontrol system against overwintering populations.

Limited transmission of turkey coronavirus in young turkeys by adult Alphitobius diaperinus (Coleoptera: Tenebrionidae).

We examined the role of lesser mealworm, Alphitobius diaperinus (Panzer), in the transmission of an enteric disease of turkeys caused by a coronavirus. Turkey coronavirus (TCV) from two sources was studied, one isolate (NC95) was embryo propagated, the second was TCV infected material from turkeys diagnosed with poult enteritis mortality syndrome (PEMS). Beetles were fed virus-infected feces mixed with chicken feed. Transmission of virus was effectively halted by surface sterilization of the beetles. Turkey poults administered beetle homogenates infected with TCV+ PEMS that had not been surface sterilized had reduced weight gains and 50% mortality. Mortality and weight gains were not effected in the NC95 group. Virus isolation procedures were performed to determine NC95 viability at varying time intervals. Beetles were dissected and the guts removed 1, 12, and 24 h after the initial viral feeding. Whole beetles were also examined for comparison. Whole beetles and beetle guts were homogenized and injected into turkey eggs for embryo propagation. Direct immunofluorescence was used to determine the presence of TCV. A. diaperinus were capable of mechanical transmission of TCV. However, only turkey embryos receiving whole beetle and beetle gut homogenates within 1 h of feeding on the virus were positive for TCV. Laboratory studies demonstrating PEMS transmission by A. diaperinus are continuing.

Identification of ice-nucleating active Pseudomonas fluorescens strains for biological control of overwintering Colorado potato beetles (Coleoptera: Chrysomelidae).

Laboratory studies were conducted to identify ice-nucleating active bacterial strains able to elevate the supercooling point, the temperature at which freezing is initiated in body fluids, of Colorado potato beetles, Leptinotarsa decemlineata (Say), and to persist in their gut. Adult beetles fed ice-nucleating active strains of Pseudomonas fluorescens, P. putida, or P. syringae at 10(6) or 10(3) bacterial cells per beetle had significantly elevated supercooling points, from -4.5 to -5.7 degrees C and from -5.2 to -6.6 degrees C, respectively, immediately after ingestion. In contrast, mean supercooling point of untreated control beetles was -9.2 degrees C. When sampled at 2 and 12 wk after ingestion, only beetles fed P. fluorescens F26-4C and 88-335 still had significantly elevated supercooling points, indicating that these strains of bacteria were retained. Furthermore, beetle supercooling points were comparable to those observed immediately after ingestion, suggesting that beetle gut conditions were favorable not only for colonization but also for expression of ice-nucleating activity by these two strains. The results obtained from exposure to a single, low dose of either bacterial strain also show that a minimum amount of inoculum is sufficient for establishment of the bacterium in the gut. Persistence of these bacteria in Colorado potato beetles long after ingestion was also confirmed using a polymerase chain reaction technique that detected ice-nucleating active bacteria by virtue of their ina genes. Application of these ice-nucleating active bacteria to elevate the supercooling point of this freeze-intolerant insect pest could significantly reduce their winter survival, thereby reducing local populations and, consequently, crop damage.

Can multiple-copy sequences of prey DNA be detected amongst the gut contents of invertebrate predators?

The first experiments to clearly demonstrate that DNA techniques might be used to detect predator-prey interactions between arthropods are reported. The accurate modelling of such interactions has depended until now upon a mixture of laboratory experiments, population monitoring and biochemical tests. The latter involve gut-content analyses, and have most recently depended upon the development of prey-specific monoclonal antibodies. Although these are excellent for detecting predation on a target prey, they are impractical for analysing the prey range of a particular predator. Molecular detection depends upon the ability of DNA to resist digestion in the predator gut and of the polymerase chain reaction (PCR) to amplify prey-specific DNA from semidigested material. As a first step, experiments using carabid beetles, Pterostichus cupreus L., as predators and mosquitoes as prey are reported. The target sequences were fully characterized multiple-copy esterase genes from two laboratory strains of Culex quinquefasciatus Say. Although DNA was extracted from homogenates of whole beetles (minus appendages), a 146 bp product could be amplified from both mosquito strains digested in the beetle gut for 28 h. The larger, 263 bp product was detectable for 28 h in one mosquito strain, but could not be amplified after 5 h from the other. Whether the beetles had eaten one mosquito or six, digested for zero or 28 h, the prey were equally detectable. Having demonstrated that shorter, multiple-copy sequences survive digestion for a considerable period in the gut of a predator, the opportunity exists to develop new detection systems for studying predation in the field.

Isolation and immunochemical characterization of neuropeptides from the midgut of the Colorado potato beetleLeptinotarsa decemlineata

The midgut of the Colorado potato beetle showed endocrine cells immunopositive to monoclonals like MAC-18, MAC-3 and polyclonals to FMRF-amide and AKH-241. Extraction and HPLC-fractionation of the midgut extracts after partial purification and characterization showed immunopositive reaction to the monoclonals and also showed myotropic activity stimulating the potato beetle gut. Molecular mass of the two active peptides isolated were 22 and 26 kDa respectively. Both these peptides could be immunocytochemically demonstrated in the brain neurosecretory cells of the red cotton bug,Dysdercus cingulatus and the castor semilooper,Achaea janata as well.

Ice-Nucleating Bacteria from the Guts of Two Sub-Antarctic Beetles,Hydromedion sparsutumandPerimylops antarcticus(Perimylopidae)

The site of ice nucleation in the freeze-tolerant, sub-Antarctic beetleHydromedion sparsutumhas been investigated. Ice+bacteria, active at above −2.0°C, were isolated from the guts of beetles and identified as a fluorescentPseudomonasspecies. Other possible sites of nucleation, including the hemolymph, were examined but had a lower activity. Ice+bacteria were isolated from mixed populations, isolated from the guts of adult beetles, and grown on nutrient agar plates and in nutrient broth. Nucleation activity of the broth culture peaked after only 2 days although the number of live cells continued to increase until day 6. These cultures were used to determine the maximum nucleation activity of a bacterial suspension in sterile distilled water (−3.4°C) and the dilution factor required to cause a 50% reduction in activity (104). The original bacterial suspension had an absorbance of 0.5 measured at 660 nm and contained 6 × 1011bacteria per milliliter. From this it is estimated that only 1 in 106bacteria possessed the highest levels of ice-nucleating activity. Other insect species, includingPerimylops antarcticus,which are found in habitats similar to that ofH. sparsutum,were examined for the presence of ice+bacteria. All contained ice-nucleating bacteria in their guts but with a lower level of activity than inH. sparsutum.

The cysteine protease activity of Colorado potato beetle ( Leptinotarsa decemlineata Say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors

High levels of protease inhibitors are induced in potato leaves by wounding. These inhibitors, when ingested by Colorado potato beetle ( Leptinotarsa decemlineata Say) larvae, induce expression of specific proteolytic activities in the gut. Induced protease activities cannot be inhibited by potato inhibitors and thus enable the insects to overcome this defence mechanism of potato plants. The induced aminopeptidase and endoproteolytic activities both have the characteristics of cysteine proteases. Twenty-one protein inhibitors of different structural types have here been examined for their ability to inhibit these activities in vitro. Members of the cystatin superfamily were found to be poor inhibitors of the induced endoproteolytic activities, except for the third domain of human kininogen, which was a fairly strong inhibitor (75% inhibition). The strongest inhibition (85%) of induced endoproteolytic activity was obtained using structurally different thyroglobulin type-1 domain-like inhibitors—equistatin and MHC class II-associated p41 invariant fragment. Experiments performed using three synthetic substrates for endoproteases gave similar results and indicate the existence of at least three different endoproteolytic enzymes resistant to potato inhibitors. The induced aminopeptidase activity can be inhibited only by stefin family of inhibitors in cystatin superfamily. In in vivo experiments, Colorado potato beetle larvae fed on equistatin-coated potato leaves were strongly retarded in their growth and almost 50% died after 4 days. This demonstrated the potential of equistatin to protect crops from insect attack.

Effect of chronic ingestion of the cysteine proteinase inhibitor, E‐64, on Colorado potato beetle gut proteinases

AbstractChronic ingestion of the highly active, specific cysteine proteinase inhibitor, E‐64, has a profound effect on Colorado potato beetle (CPB) larval growth, development and survival, as well as on adult fecundity. However, the number of insects surviving to the adult stage did not decrease below 26% with increasing E‐64 concentration above 1.5 μg E‐64 cm−2 leaf surface. The development time to the pupal stage was increased from 13 days, when larvae were reared on control leaves, to 21 days at a concentration of 1.5 μg E‐64 cm−2. The most significant effect of dietary E‐64 was on adult fecundity, with mated females reared on untreated leaves laying an average 62 ± 5.7 eggs daily in the first 10 days, and those maintained on 0.5 μg E‐64 cm−2, laying only 16 ± 2.4 eggs day−1. Females given 1 μg E‐64 cm−2 laid few if any eggs, but started producing egg masses as large as control insects about 5 days after being switched to control leaves. These effects on the insect life cycle were directly related to the degree of inhibition of cysteine proteinase activity in gut extracts. The general proteinase activity in control extracts was 6.5 ± 0.16 units min−1 mg gut−1, which decreased to 1.9 ± 0.16 in guts of insects reared on 1 μg E‐64 cm−2. The proportion of proteinase activity inhibitable by E‐64 decreased from 66% in control guts to 10‐15% in guts from larvae reared on 1 μg E‐64 cm−2. The aspartate proteinase inhibitor, pepstatin, decreased proteinase activity by 35% in control guts. There was no induction of pepstatin‐inhibitable proteinases in response to inhibition by E‐64, and no inhibition of gut enzyme activity by soybean trypsin inhibitor from larvae fed any of the E‐64 concentrations. This study demonstrates that proteinase levels must be significantly reduced to have a pronounced effect on larval growth and survival, while fecundity of mated females is affected by lower concentrations of inhibitor. It also suggests that the CPB may be a difficult pest to control using a more specific, plant‐derived cysteine proteinase inhibitor, such as oryzacystatin.

New Pest Control Strategies and Effects on Pollination of Cucumbers

Cucumbers are susceptible to the bacterial wilt organism that overwinters in the gut of cucumber beetles. This disease is transmitted in feces via open feeding wounds and plugs xylem vessels of water conductive tissues. Insecticides can be applied to control cucumber beetles. Adios, a semiochemical bait impregnated with cucurbitacin is combined with the insecticide carbaryl, which can be applied after plant emergence to control cucumber beetles. However, the method of application for giving the maximum control is unknown; thus, this was the purpose of this project. This study evaluates the rate of application, number of applications, methods of application using pressure and airblast sprays, and compares two Adios formulations. Also studied were the effects of Adios on bee fertilization and the quality of the fruit, since carbaryl is toxic to bees, and therefore can affect pollination. Adios was also compared to a foliar insecticide, Asana.

Insect fungal symbionts: A promising source of detoxifying enzymes

Many species of insects cultivate, inoculate, or contain symbiotic fungi. Insects feed on plant materials that contain plant-produced defensive toxins, or are exposed to insecticides or other pesticides when they become economically important pests. Therefore, it is likely that the symbiotic fungi are also exposed to these toxins and may actually contribute to detoxification of these compounds. Fungi associated with bark beetles, ambrosia beetles, termites, leaf-cutting ants, long-horned beetles, wood wasps, and drug store beetles can variously metabolize/detoxify tannins, lignins, terpenes, esters, chlorinated hydrocarbons, and other toxins. The fungi (Attamyces) cultivated by the ants and the yeast (Symbiotaphrina) contained in the cigarette beetle gut appear to have broad-spectrum detoxifying abilities. The present limiting factor for using many of these fungi for large scale detoxification of, for example, contaminated soils or agricultural commodities is their slow growth rate, but conventional strain selection techniques or biotechnological approaches should overcome this problem.

The Role of Trichosporon cutaneum in Eliciting Aggregation Behavior in Cotinis nitida (Coleoptera: Scarabaeidae)

Aggregation behavior was elicited in both sexes of the green June beetle, Cotinis nitida (L.), when green June beetle males fed on peach slices inoculated with one yeast species, Trichosporon cutaneum (de Beurm. Gougerot et Voucher) Ota. Peach puree monocultures were made by inoculating sterile peach puree with one of three yeasts ( Aureobasidium pullulans (de Bary) Arnaud, Candida krusei (Castellani) Berkhout, C. tropicalis (Castellani) Berkhout), which were all endemic flora isolated from the feces and gut of green June beetles collected from feeding aggregations on unsterile peaches in the field (natural bait). In a field bioassay, peach puree monocultures of C. tropicalis (in experiment A) and C. krusei (in experiment B), both without beetle feeding, were significantly more attractive ( P < 0.05) to both sexes of green June beetles than sterile peach puree with or without beetle feeding, but all peach purees were significantly less attractive than the most attractive natural bait. In experiment C, peach slices were inoculated with either of two yeasts (biovars A or B of T. cutaneum ), which were the predominant isolates from the gut of laboratory-reared green June beetles fed on unsterile peaches. These monocultures of T. cutaneum on peach slices without beetle feeding were relatively as attractive as monoculture-inoculated peach puree alone containing one of the three other yeasts noted above. But when laboratory-reared green June beetle males previously fed on the T. cutaneum -inoculated peach slices were included with these peach slices, the bait containing biovar A was as attractive as the natural bait. Although the bait of laboratory-reared green June beetle males feeding on peach slices inoculated with biovar B was significantly less attractive ( P < 0.05) than the natural bait, it was Similarly attractive to the laboratory-reared green June beetle males feeding on the biovar A-inoculated peach slices. The laboratory-reared green June beetles required a period of 1 d or more of feeding on yeast-inoculated fruit to achieve peak production of aggregation semiochemical.

Development of a Yeast Flora in the Adult Green June Beetle (Cotinis nitida, Scarabaeidae)

Adult green June beetle guts contained no yeasts prior to the beetles' emergence from pupation. As beetles emerged from soil and fed, a gut yeast flora developed, increasing in size and changing in character with feeding duration from a largely fermentative flora to one in which obligately respiratory yeasts were prominent. Newly emerged adults after 24 hour feeding contained ca 103 colony-forming units of yeasts per milligram of gut; longer feeding on peach slices yielded ca 2 × 107 colony-forming units per milligram of gut. Aureobasidium pullulans, Candida guilliermondii, C. krusei, C. sake, C. tropicalis, Cryptococcus albidus var. albidus, Debaryomyces hansenii (3 biovars), Hanseniaspora uvarum, Hansenula californica, Rhodotorula glutinis, R. rubra, and Trichosporon cutaneum (2 biovars) were identified from gut and fecal samples. Peaches which had been fed upon acquired the major part of their yeast flora from the feeding beetles. Trichosporon cutaneum was positively selected in both beetle gut and peach slice environments; this species is predicted to be involved in production of semiochemical(s) responsible for beetle feeding aggregation enhancement.

Development of a Yeast Flora in The Adult Green June Beetle (Cotinis Nitida, Scarabaeidae)

Adult green June beetle guts contained no yeasts prior to the beetles' emergence from pupation. As beetles emerged from soil and fed, a gut yeast flora developed, increasing in size and changing in character with feeding duration from a largely fermentative flora to one in which obligately respiratory yeasts were prominent. Newly emerged adults after 24 hour feeding contained ca 103 colony-forming units of yeasts per milligram of gut; longer feeding on peach slices yielded cal x 107 colony-forming units per milligram of gut. Aureobasidium pullulans, Candida guilliermondii, C. krusei, C. sake, C. tropicalis, Cryptococcus albidus var. albidus, Debaryomyces hansenii (3 biovars), Hanseniospora uvarum, Hansenula californica, Rhodotorula glutinis, R. rubra, and Trichosporon cutaneum (2 biovars) were identified from gut and fecal samples. Peaches which had been fed upon acquired the major part of their yeast flora from the feeding beetles. Trichosporon cutaneum was positively selected in both beetle gut and peach slice environments; this species is predicted to be involved in production of semiochemical(s) responsible for beetle feeding aggregation enhancement.

Plant cells which aid in pollen digestion within a beetle's gut.

The peach palm, Bactris gasipaes H.B.K., in Costa Rica, possesses unusual trichomes on the inflorescence epidermal surface. Certain cells of the trichome possess a thick, highly lignified cell wall and are consumed by the beetle Cyclocephala amazona L. before it ingests pollen from the same inflorescence. Chemical analyses show the trichome to possess no nutritive value. The thick-walled trichome cells pass intact through the beetle's digestive system, while ingested pollen is crushed. We suggest that the specialized plant cells function as gastroliths in the beetle's digestive tract.

Parasitic Microorganisms of Japanese Beetle (Coleoptera: Scarabaeidae) and Associated Scarab Larvae in Connecticut Soils

The parasites of second- and third-instar Japanese beetles, Popillia japonica Newman, and associated scarab larvae were identified in turf samples from 49 locations in Connecticut during the fall of 1986. Four of the seven species of scarab larvae encountered were introduced and accounted for 91% of the sample population. Recovered parasites included three species of protozoa, two bacteria, a rickettsia, and a fungus. The most common protozoa, cephaline eugregarines, were found in the gut of Japanese beetles from 42 locations, and in four other host species. A microsporidium, Ovavesicula popilliae Andreadis and Hanula, was found in Japanese beetles from 34 sites. Overall, 25% of the larvae were infected, but prevalence was 80–90% in some locations. An Adelina sp. infecting 19% of the Asiatic garden beetles, Maladera castanea (Arrow), was found at six locations and in two other scarab species. This is the first record of Adelina sp. in these hosts. The bacteria Bacillus popilliae Dutky and B. lentimorbus Dutky and a rickettsia, Rickettsiella popilliae (Dutky and Gooden) Philip, were also recovered from grubs. R. popilliae was recovered from five species. Two of the infected species, the Asiatic garden beetle and the European chafer, Rhizotrogus majalis (Razoumowsky), are new records as natural hosts for this pathogen. The incidence of B. popilliae (3.5%) was comparable with previous reports from Connecticut. The fungus, Metarhizium anisopliae (Metch.), infected 1.2% of the Japanese beetles.