Redescription and Life History of Contortylenchus brevicomi,a Parasite of the Southern Pine Beetle Dendroctonus frontalis

Larval and adult life stages are described for Contortylenchus brevicomi (Massey) Rühm parasitizing a Mississippi population of Dendroctonus frontalis, the southern pine beetle. Fourth-stage larvae and free-living adult females of this species are identified and described for the first time. The life cycle of C. brevicomi can be reconstructed from this study. The adult female nematode lays eggs in a mature beetle. Larval development progresses within the hemocoel until fourth-stage larvae exit the host. Mating occurs in beetle galleries and only females enter an immature beetle host.     

Fighting while Parasitized: Can Nematode Infections Affect the Outcome of Staged Combat in Beetles?

The effects of non-lethal parasites may be felt most strongly when hosts engage in intense, energy-demanding behaviors. One such behavior is fighting with conspecifics, which is common among territorial animals, including many beetle species. We examined the effects of parasites on the fighting ability of a saproxylic beetle, the horned passalus (Odontotaenius disjunctus, Family: Passalidae), which is host to a non-lethal nematode, Chondronema passali. We pitted pairs of randomly-chosen (but equally-weighted) beetles against each other in a small arena and determined the winner and aggression level of fights. Then we examined beetles for the presence, and severity of nematode infections. There was a non-significant tendency (p = 0.065) for the frequency of wins, losses and draws to differ between beetles with and without C. passali; non-parasitized individuals (n = 104) won 47% of their fights while those with the parasite (n = 88) won 34%, a 13% difference in wins. The number of nematodes in a beetle affected the outcome of fights between infected and uninfected individuals in an unexpected fashion: fighting ability was lowest in beetles with the lowest (p = 0.033), not highest (p = 0.266), nematode burdens. Within-fight aggression was highest when both beetles were uninfected and lowest when both were infected (p = 0.034). Collectively, these results suggest the nematode parasite, C. passali, is associated with a modest reduction in fighting ability in horned passalus beetles, consistent with the idea that parasitized beetles have lower energy available for fighting. This study adds to a small but growing body of evidence showing how parasites negatively influence fighting behavior in animals.     

Transmission of Bursaphelenchus xylophilus through Oviposition Wounds of Monochamm carolinensis (Coleoptera: Cerambycidae)

Transmission of pinewood nematode through Monochamus carolinensis oviposition wounds was documented. Nematode transmission was measured as the average number of nematodes isolated per oviposition wound excavated and also as the percentage of oviposition wounds from which nematodes were isolated. The influence of three factors that might affect nematode transmission was investigated: age of the beetle vector, number of nematodes carried per beetle, and egg deposition in the oviposition wound. Only the number of nematodes carried by the beetle was found to have a significant effect on transmission. Nematodes were transmitted more frequently and in slightly greater numbers by beetles carrying more nematodes. The influence of pinewood on nematode exit from beetles were investigated by comparing nematode exit from beetles placed over pine chips with those placed over distilled water. Nematodes exited in greater numbers and at a higher frequency from beetles over pine chips than from beetles over distilled water. Apparently, the nematodes are able to detect a factor from the pine chips that promotes their exit from the beetles.     

Effect of Monochamus carolinensis on the Life History of the Pinewood Nematode,Bursaphelenchus xylophilus

The development of Bursaphelenchus xylophilus in pine wood infested with and free of Monochamus carolinensis was investigated. Formation of third-stage dispersal juveniles occurred in the presence and absence of pine sawyer beetles. The proportion of third-stage dispersal juveniles in the total nematode population was negatively correlated with moisture content of the wood. Formation of nematode dauer juveniles was dependent on the presence of the pine sawyer beetle. Dauer juveniles were present in 3 of 315 wood samples taken from non-beetle-infested Scots pine bolts and 81 of 311 samples taken from beetle-infested bolts. Nematode densities were greater in wood samples taken adjacent to insect larvae, pupae, and teneral adults compared with samples taken from areas void of insect activity. Nematodes recovered from beetle larvae, pupae, and teneral adults were mostly fourth-stage dauer juveniles, although some third-stage dispersal juveniles were also recovered. Dauer juvenile density was highest on teneral adult beetles.     

Broadscale Specificity in a Bark Beetle–Fungal Symbiosis: a Spatio-temporal Analysis of the Mycangial Fungi of the Western Pine Beetle

Whether and how mutualisms are maintained through ecological and evolutionary time is a seldom studied aspect of bark beetle–fungal symbioses. All bark beetles are associated with fungi and some species have evolved structures for transporting their symbiotic partners. However, the fungal assemblages and specificity in these symbioses are not well known. To determine the distribution of fungi associated with the mycangia of the western pine beetle (Dendroctonus brevicomis), we collected beetles from across the insect’s geographic range including multiple genetically distinct populations. Two fungi, Entomocorticium sp. B and Ceratocystiopsis brevicomi, were isolated from the mycangia of beetles from all locations. Repeated sampling at two sites in Montana found that Entomocorticium sp. B was the most prevalent fungus throughout the beetle’s flight season, and that females carrying that fungus were on average larger than females carrying C. brevicomi. We present evidence that throughout the flight season, over broad geographic distances, and among genetically distinct populations of beetle, the western pine beetle is associated with the same two species of fungi. In addition, we provide evidence that one fungal species is associated with larger adult beetles and therefore might provide greater benefit during beetle development. The importance and maintenance of this bark beetle–fungus interaction is discussed.     

Reproduction of Meloidogyne javanica on Plant Roots Genetically Transformed by Agrobacterium rhizogenes

Reproduction of Meloidogyne javanica was compared on several Agrobacterium rhizogenes-transformed root cultures under monoxenic conditions. M. javanica reproduced on all transformed roots tested; however, more females and eggs were obtained on potato and South Australian Early Dwarf Red tomato than on bindweed, Tropic tomato, lima bean, or carrot. Roots that grew at moderate rates into the agar and produced many secondary roots supported the highest reproduction. Numbers of females produced in cultures of transformed potato roots increased with increasing nematode inoculum levels, whether inoculum was dispersed eggs or juveniles. Females appeared smaller, produced fewer eggs, and were found in coalesced galls at the higher inoculum levels. The ratio between the final and initial population decreased sharply as the juvenile inoculum increased. The second-stage juvenile was preferred to dispersed eggs or egg masses for inoculation of tissue culture systems because quantity and viability of inoculum were easily assessed. Meloidogyne javanica reared on transformed root cultures were able to complete their life cycles on new transformed root cultures or greenhouse tomato plants.     

Parasites and Dung Beetles as Ecosystem Engineers in a Forest Ecosystem

Dung beetles serve as the intermediate host for Streptopharagus pigmentatus, a nematode parasite that infects an old world primate, the Japanese Macaque (Macaca fuscata). This study compares the behaviors of infected and uninfected beetles in both transmission dynamics and the ecological role of the parasite. The results suggest that parasitism does not alter the beetle’s use of shelter or choice of substrate on Yakushima Island, Japan. However, infected beetles consume significantly less feces. Dung beetles remove the majority of fecal material in this forest ecosystem, eliminating breeding grounds for many insect pests and burying nutrients that are essential for plant health. Thus, the nematode parasite S. pigmentatus, by altering its host’s behavior, changes the availability of fecal resources to both plant and animal communities and should therefore be classified as an ecosystem engineer.     

Inhibitory Effect of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) on B. xylophilus Boarding Adult Monochamus alternatus (Coleoptera: Cerambycidae)

Inhibitory effects of Bursaphelenchus mucronatus on the number of B. xylophilus carried by an adult Monochamus alternatus were investigated using artificial pupal chambers. When pupal chambers were infested with either B. xylophilus or B. mucronatus, the load of B. xylophilus onto the beetle was greater (P < 0.001) than that of B. mucronatus. However, within the pupal chamber there was no difference in the abundance of the third-stage dispersal juveniles, which would molt to the fourth-stage dispersal juveniles to board beetles. The nematode load on beetles that emerged from pupal chambers infested with both Bursaphelenchus species was smaller (P = 0.015) than that of beetles with B. xylophilus alone but greater (P < 0.001) than that of beetles with B. mucronatus alone, suggesting an inhibitory effect of B. mucronatus. As a result of this study, the rate of inhibition of B. mucronatus on molting of third-stage dispersal juveniles of B. xylophilus to fourth-stage dispersal juveniles was 0.65, which resulted in great inhibition on boarding beetles at a rate of 0.7.     

Temporal Pattern of Pinewood Nematode Exit from the Insect Vector Monochamus carolinensis

Laboratory-reared Monochamus carolinensis (Olivier) were used to study the temporal pattern of pinewood nematode dauer larval exit from this beetle vector. Exit rates of dauer larvae were determined by comparing the mean number of dauer larvae carried by adult beetles 0, 7, 14, or 21 days after emergence from the log in which they developed. Density of dauer larvae was highest in beetles on the day of their emergence and dropped slowly through the subsequent age classes. The rate of nematode exit was low during the first week (4.5%) and higher during weeks 2 (20.5%) and 3 (13.1%). A total of 38.1% of the initial dauer larvae exited the beetles during the 3-week observation period.     

Effect of Meloidogyne incognita and Importance of the Inoculum on the Yield of Eggplant

The relationship between population densities of race 1 of Meloidogyne incognita and yield of eggplant was studied. Microplots were infested with finely chopped nematode-infected pepper roots to give population densities of 0, 0.062, 0.125, 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, and 128 eggs and juveniles/cm³ soil. Both plant growth and yield were suppressed by the nematode. A tolerance limit of 0.054 eggs and juveniles/cm³ soil and a minimum relative yield of 0.05 at four or more eggs and juveniles/cm³ soil were derived by fitting the data with the equation y = m + (1 - m)z^P⁻T. Maximum nematode reproduction rate was 12,300. Hatch of eggs from egg masses in water or from sodium hypochlorite dissolved egg masses was similar (41% and 39%), but egg viability was significantly greater from egg masses in water (58%) than from sodium hypochlorite dissolved egg masses (12%) after 4 weeks. Greater numbers of nematodes were collected from roots of tomatoes from soil infested with entire egg masses than from tomato roots from soil infested with egg masses dissolved by sodium hypochlorite.     

Transmission of the Pinewood Nematode, Bursaphelenchus xylophilus,to Slash Pine Trees and Log Bolts by a Cerambycid Beetle,Monochamus titillator, in Florida

Field-collected adults of the southern pine sawyer, Monochamus titillator (F.) (Coleoptera: Cerambycidae), naturally infested with fourth-stage juveniles (dauerlarvae) of the pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer, 1934) Nickle, 1970, were maturation fed on excised shoots of typical slash pine, Pinus elliottii Engelm. var elliottii, for 21 days. During August 1981, a male and female adult beetle were held in a sleeve cage placed on the terminal of a side branch of each of seven replicate, healthy 10-year-old slash pine trees. All seven branch terminals showed evidence of beetle feeding on the bark after 1 week, and pinewood nematodes were present in wood samples taken near these feeding sites. Four of the seven trees showed wilt symptoms in 4-6 weeks and died about 9 weeks after beetle feeding. Pinewood nematodes were recovered from the roots and trunks of the dead trees. Each of seven replicate slash pine log bolts was enclosed in a jar with a pair of the same beetles used in the sleeve cages. After 1 week, wood underlying beetle oviposition sites in the bark of all replicate log bolts was infested with the pinewood nematode.     

Effects of the bark beetle nematode, Contortylenchus reversus, on gallery construction,fecundity, and egg viability of the Douglas Fir beetle, Dendroctonus pseudotsugae (Coleoptera: Scolytidae)

Healthy and Contortylenchus reversus infected Douglas Fir beetles were colonized in logs in the laboratory. At 7, 14, and 23 days after introduction of the beetles into the logs, the bark was removed from some of the logs and the length and shape of the primary gallery, the number of eggs laid, and the egg viability were determined for each pair of parent beetles. Parasitism caused a 25, 28, and 27% reduction in the length of the primary egg gallery built by the infected female at 7, 14, and 23 days, respectively, after the start of colonization. In addition, the number of eggs laid by the infected females by 7, 14, and 23 days after the start of colonization were 50, 33, and 45%, respectively, lower than that of healthy female beetles. Despite the differences in gallery length between control and infected females, linear regression analysis showed that the rates of gallery construction were not significantly different. However, infected female beetles showed significantly lower rates of egg-laying than did healthy female beetles. Nematode parasitism did not affect gallery shape and egg viability of female Douglas Fir beetles. Mating of healthy female beetles with infected males affected neither the gallery length nor the fecundity of the uninfected females.     

Laboratory study of conspecific avoidance by host-colonizingDendroctonus frontalis Zimm. (Coleoptera: Scolytidae)

Bark/phloem disk sandwiches (100 cm²) were colonized with one to six mating pairs of the southern pine beetle, Dendroctonus frontalis Zimm., to observe gallery construction and evaluate if avoidance behavior occurs between beetles. Frequencies of turn aways, touches, and crosses (three characteristics used to describe conspecific interaction) all increased significantly with beetle density. A threshold of three mating pairs within the 100- cm² phloem area was the density at which avoidance behavior became evident. At a threshold of five mating pairs the number of gallery crossings was significantly greater than at lower densities. Single and paired female beetles introduced into artificial galleries crossed empty neighboring galleries significantly more often than when neighboring galleries were occupied with other D. frontalis. The data suggest that D. frontalis females can regulate gallery spacing within the host through their ability to detect nearby beetles and their galleries. Possible spacing mechanisms are discussed.     

Newly Discovered Transmission Pathway of Bursaphelenchus xylophilus from Males of the Beetle Monochamus alternatus to Pinus densiflora Trees via Oviposition Wounds

The transmission of Bursaphelenchus xylophilus from Monochamus alternatus males to Pinus densiflora trees via oviposition wounds has been determined. Nematode-infested males, with mandibles fixed experimentally to prevent feeding, were placed for 48 hours with pine bolts containing oviposition wounds that had been made by nematode-free females. After removal of the nematode-infested males, the pine bolts were held for 1 month and then examined for the presence of nematodes. Reproducing nematode populations were recovered from pine bolts that were exposed to male beetles carrying a high number of nematodes. No reproducing nematode population could be recovered from pine bolts exposed to beetles with a small number of nematodes. Nematode reproduction in the pine bolts was not related to the number of oviposition wounds per bolt. Fourth-stage dispersal B. xylophilus juveniles, collected from beetle body surfaces, were inoculated on pine bolt bark 0, 5, 10, and 15 cm away from a single artificial, small hole. These dauer juveniles successfully entered some bolts. The probability of successful nematode reproduction decreased with increased distance between inoculation point and artificial hole. The results indicated that B. xylophilus can move a significant distance to oviposition wounds along the bark surface and enter a tree via the wounds. The new transmission pathway is considered important for the nematode to persist in pine forests such as in North America where pine wilt disease does not occur.     

Transmission of Pinewood Nematode Through Feeding Wounds of Monochamus carolinensis (Coleoptera: Cerambycidae)

Transmission of pinewood nematode, Bursaphelenchus xylophilus, to mature, field grown Scots pines through feeding wounds of Monochamus carolinensis was investigated by caging nematode-infested beetles on pine branches for 24 hours. Nematodes were transmitted to 31 of 64 branches. Frequency of successful transmission was independent of the sex of the beetle but dependent upon beetle age. Transmission frequencies were highest for beetles 2, 3, and 4 weeks after emergence as adults. The number of nematodes transmitted per branch was low and did not differ between beetle sexes or among beetle age categories. The number of nematodes extracted per branch was correlated with the number of nematodes carried per beetle but was not correlated with the feeding area on the branch.     

Ecological Study of Nematode Parasitism in Ips Beetles from California and Idaho

Nematodes found in Ips paraconfusus from ponderosa pine in California were an undescribed species of Parasitaphelenchus, Contortylenchus elongatus, C. reversus, and C. brevicomi. C. elongatus, the most commonly found contortylenchid, was present in 98.2% of the contortylenchid-parasitized beetles. Only one nematode parasite of the gut, a Parasitorhabditis sp., was isolated. Although significant differences in parasitism were observed, they were by collection sites, rather than by elevation or bole sources (slash or standing). Significant changes in parasitism between fall and spring collections were observed but not at every site. Nematode parasitism in the F₁ generation of I. paraconfusus by Parasitaphelenchus, Contortylenchus, or Parasitorhabditis increased or decreased from the parent generation depending upon the experiment.Nematode parasites from I. pini included an undescribed Parasitaphelenchus sp., two undescribed Contortylenchus spp., C. reversus and Parasitylenchus (= Neoparasitylenchus) ovarius from the hemocel, and Parasitorhabditis ipini from the gut. Parasitaphelenchus sp. was found in 99% and 45.3% of the beetles from Idaho and California, respectively. Of the 1,000 I. pini from Idaho and California, 157 were parasitized by the contortylenchid species or P. ovarius.     

Horizontal Transmission of Bursaphelenchus xylophilus between Sexes of Monochamus alternatus

Four experiments were conducted using nematode-infested and nematode-free adults of the cerambycid beetle, Monochamus alternatus, to determine horizontal transmission pathways of Bursaphelenchus xylophilus. When nematode-infested beetles of one sex and nematode-free beetles of the opposite sex were paired in containers for 48 or 72 hours, the number of nematodes carried by nematode-free beetles tended to increase with increased number of nematodes carried by nematode-infested beetles. The nematodes acquired by "nematode-free" beetles could be transmitted to pine. A female beetle that received 13 nematodes from a male transmitted one nematode to a Pinus densiflora bolt via an oviposition wound. When the nematode-infested and nematode-free beetles were observed continuously, it was observed that the number of nematodes carried by nematode-free beetles at the end of the first sexual mounting increased as the number of nematodes carried by nematode-infested beetles just before mounting increased. The number of nematodes transferred to nematode-free beetles was positively related to duration time of mounting. There was no difference in transmission efficacy between male-to-female transmission and female-to-male transmission. The horizontal transmission pathways are discussed relative to the persistence of B. xylophilus in resistant pine forests and the control of pine wilt disease.     

Experimental evidence of bark beetle adaptation to a fungal symbiont

The importance of symbiotic microbes to insects cannot be overstated; however, we have a poor understanding of the evolutionary processes that shape most insect–microbe interactions. Many bark beetle (Coleoptera: Curculionidae, Scolytinae) species are involved in what have been described as obligate mutualisms with symbiotic fungi. Beetles benefit through supplementing their nutrient‐poor diet with fungi and the fungi benefit through gaining transportation to resources. However, only a few beetle–fungal symbioses have been experimentally manipulated to test whether the relationship is obligate. Furthermore, none have tested for adaptation of beetles to their specific symbionts, one of the requirements for coevolution. We experimentally manipulated the western pine beetle–fungus symbiosis to determine whether the beetle is obligately dependent upon fungi and to test for fine‐scale adaptation of the beetle to one of its symbiotic fungi, Entomocorticium sp. B. We reared beetles from a single population with either a natal isolate of E. sp. B (isolated from the same population from which the beetles originated), a non‐natal isolate (a genetically divergent isolate from a geographically distant beetle population), or with no fungi. We found that fungi were crucial for the successful development of western pine beetles. We also found no significant difference in the effects of the natal and non‐natal isolate on beetle fitness parameters. However, brood adult beetles failed to incorporate the non‐natal fungus into their fungal transport structure (mycangium) indicating adaption by the beetle to particular genotypes of symbiotic fungi. Our results suggest that beetle–fungus mutualisms and symbiont fidelity may be maintained via an undescribed recognition mechanism of the beetles for particular symbionts that may promote particular associations through time.     

Effects of Fluctuating Temperatures and Different Host Plants on Development of Pasteuria penetrans in Meloidogyne javanica

Greenhouse and growth room experiments were conducted to investigate the effect of host plant in relation to different nematode inoculum levels, and temperature fluctuations on the development of Pasteuria penetrans. Host plant affected the development of P. penetrans indirectly through its effect on nematode development. Endospores collected from Meloidogyne javanica females reared on different hosts did not show any differences in subsequent attachment and infectivity. The numbers of endospores produced per infected female were reduced with increasing numbers of females parasitizing okra and tomato roots. Fluctuating temperatures retarded the development of P. penetrans. The life cycle of the parasite was completed faster at approximately constant temperatures close to 30 °C than when the temperature fluctuated away from 30 °C. The temperature of irrigation water did not affect the duration of life cycle of P. penetrans.     

A Nondestructive Method of Determining Bursaphelenchus xylophilus Infestation of Monochamus spp. Vectors

Pine wilt is caused by the nematode Bursaphelenchus xylophilus, which is transported to host trees in the trachea of Monochamus spp. (Coleoptera: Cerambycidae). The study of the relationship between the nematode and its beetle vectors has been hampered by the inability to estimate nematode presence or density within live beetles. This report describes a rapid method for estimating nematode load within live M. carolinensis and M. alternatus by visual examination of the atrium of the first abdominal spiracle. Visual estimates of nematode numbers correlated highly with actual nematode numbers. This method is a timesaving technique for determining relative numbers of B. xylophilus in pine wilt research.