Phylogenetic analyses of Reticulitermes (Blattodea: Rhinotermitidae) from California and other western states: multiple genes confirm undescribed species identified by cuticular hydrocarbons.

Subterranean termites in the genus Reticulitermes Holmgren 1913 are among the most economically important wood-destroying pests in the western United States. Yet, there remains uncertainty regarding the taxonomy and biology of the species in this genus. The 2 species described as having distributions in this region are the western subterranean termite, Reticulitermes hesperus Banks, and the arid land subterranean termite, Reticulitermes tibialis Banks. Taxonomic studies utilizing cuticular hydrocarbon (CHC) profiles, agonistic behavior, flight phenology, and mitochondrial DNA (mtDNA) suggested that R. hesperus is a species complex comprised of 2 or more sympatric, yet reproductively isolated species. To further delineate these taxa, we examined multiple genes from samples of Reticulitermes collected in the western United States. Alates collected after recent spring and fall mating flights, as well as previously collected workers, were subjected to CHC phenotyping and DNA sequence analyses that targeted mitochondrial cytochrome oxidase subunit II (COII), mitochondrial 16S rRNA, and nuclear Internal Transcribed Spacer 1 and 2 (ITS1 and 2). Phylogenetic analyses conducted also included published sequences of other putative western Reticulitermes species. Results suggest that at least 5 species of Reticulitermes may be present in California and that Reticulitermes in Arizona consistently group into multiple clades, including samples previously identified as R. tibialis in a sister clade. These analyses further support the species status of qualitatively different CHC phenotypes and that alates swarming in spring vs. fall are reproductively isolated species.

Consistency of Cuticular Hydrocarbon Mixtures of Five Reticulitermes (Blattodea: Rhinotermitidae) Taxa From Northern California: Similarity Among Colonies and Seasonal Variation.

Cuticular hydrocarbon (CHC) mixtures from workers of five distinct CHC phenotypes of Reticulitermes Holmgren 1913 from two locations in northern California were examined from monthly collections taken over a 3-yr period. The objectives of this study were (1) to identify and quantify variations of the CHCs of multiple colonies of each of these phenotypes (= species or subspecies) to demonstrate consistency, (2) to assess the potential of CHC mixtures to separate or identify colonies within each phenotype, and (3) to detect any temporal changes in each of the hydrocarbons in the CHC mixtures. Nonmetric multidimensional scaling of all CHC mixtures of all samples collected at both locations separated the samples into five clearly visible, different groups of CHC phenotypes (taxa or species) of Reticulitermes. The degree of variability of the CHC mixtures among colonies of each phenotype was such that nonmetric multidimensional scaling did not separate or identify colonies. Strong seasonal fluctuations were evident in some of the CHCs of all five phenotypes and were significantly consistent with a sine curve. Maximum proportions of seasonal CHCs within a phenotype occurred in all seasons of the year but occurred mostly in the winter and summer. In general, the CHCs displaying maximum values in the winter were short-chained (C23-C27) methyl-branched alkanes, whereas the CHCs displaying maximum values in the summer were long-chained (C35-C43) methyl-branched alkanes, which likely influences water retention. These consistent chemical fingerprints are probably responsible for inter-phenotype recognition patterns and are thus useful for chemical taxonomy.

Trapping Failure Leads to Discovery of Potent Semiochemical Repellent for the Walnut Twig Beetle.

The walnut twig beetle, Pityophthorus juglandis Blackman, and its associated fungal pathogen that causes thousand cankers disease, currently threaten the viability of walnut trees across much of North America. During a 2011 assessment of seasonal flight patterns of P. juglandis with yellow sticky traps baited with the male-produced aggregation pheromone component, 3-methyl-2-buten-1-ol, dramatically reduced catches were recorded when Tree Tanglefoot adhesive was used to coat the traps. In summer 2011, two trap adhesives were tested for potential repellency against P. juglandis in a field trapping bioassay. SuperQ extracts of volatiles from the most repellent adhesive were analyzed by gas chromatography-mass spectrometry, and limonene and α-pinene were identified as predominant components. In field-based, trapping experiments both enantiomers of limonene at a release rate of ~700 mg/d conferred 91-99% reduction in trap catches of P. juglandis to pheromone-baited traps. (+)- and (‒)-α-Pinene reduced trap catch by 40 and 53%, respectively, at the highest release rate tested. While a combination of R-(+)-limonene and (+)-α-pinene resulted in a 97% reduction in the number of P. juglandis caught, the combination did not consistently result in greater flight trap catch reduction than individual limonene enantiomers. The repellent effect of limonene may be valuable in the development of a semiochemical-based tool for management of P. juglandis and thousand cankers disease.

Comparative morphometric and chemical analyses of phenotypes of two invasive ambrosia beetles (Euwallacea spp.) in the United States.

The polyphagous shot hole borer (PSHB), Euwallacea sp., was first detected in 2003 in Los Angeles County, California, USA. Recently, this invasive species has become a major pest of many hardwood trees in urban and wildland forests throughout southern California. PSHB is nearly identical in morphology and life history to the tea shot hole borer (TSHB), Euwallacea fornicatus, an invasive pest of hardwoods in Florida, USA and many other parts of the world. However, molecular studies have suggested that the taxa are different species. We conducted morphometric and chemical analyses of the phenotypes of Euwallacea sp. collected in southern California (Los Angeles County) and E. fornicatus collected in Florida (Miami-Dade County). Our analyses indicated that PSHB has 3 larval instars. The third larval instar was separated from the first 2 instars by head capsule width with 0 probability of misclassification. The body length, head width, and pronotal width of PSHB adult males were significantly less than those of females. Head width and pronotal width of female PSHB were significantly less than those of female TSHB. In contrast, body length, and ratio of body length to pronotal width of female PSHB were significantly greater than those of female TSHB. However, females of these 2 species could not be separated completely by these 4 measurements because of the overlapping ranges. Cuticular hydrocarbons detected in both species were exclusively alkanes (i.e., n-alkanes, monomethylalkanes, dimethylalkanes, and trimethylalkanes). Cuticular hydrocarbon profiles of PSHB males and females were similar, but they both differed from that of TSHB females. Cuticular hydrocarbons of PSHB were predominantly internally branched dimethylalkanes with backbones of 31 and 33 carbons, whereas cuticular hydrocarbons of TSHB females were dominated by internally branched monomethylalkanes and dimethylalkanes with backbones of 28 and 29 carbons. Multiple compounds within these classes appear to be diagnostic for PSHB and TSHB, respectively.

Quantitative changes in hydrocarbons over time in fecal pellets of Incisitermes minor may predict whether colonies are alive or dead.

Hydrocarbon mixtures extracted from fecal pellets of drywood termites are species-specific and can be characterized to identify the termites responsible for damage, even when termites are no longer present or are unable to be recovered easily. In structures infested by drywood termites, it is common to find fecal pellets, but difficult to sample termites from the wood. When fecal pellets appear after remedial treatment of a structure, it is difficult to determine whether this indicates that termites in the structure are still alive and active or not. We examined the hydrocarbon composition of workers, alates, and soldiers of Incisitermes minor (Hagen) (family Kalotermitidae) and of fecal pellets of workers. Hydrocarbons were qualitatively similar among castes and pellets. Fecal pellets that were aged for periods of 0, 30, 90, and 365 days after collection were qualitatively similar across all time periods, however, the relative quantities of certain individual hydrocarbons changed over time, with 19 of the 73 hydrocarbon peaks relatively increasing or decreasing. When the sums of the positive and negative slopes of these 19 hydrocarbons were indexed, they produced a highly significant linear correlation (R² =0.89). Consequently, the quantitative differences of these hydrocarbons peaks can be used to determine the age of worker fecal pellets, and thus help determine whether the colony that produced them is alive or dead.

Response of Reticulitermes hesperus (Isoptera: Rhinotermitidae) colonies to baiting with lufenuron in northern California.

The objective of this study was to evaluate lufenuron termite bait (1,500 ppm) for the elimination of colonies of Reticulitermes hesperus Banks (Isoptera: Rhinotermitidae). Dispersion of colonies in six baited and six unbaited sites near Placerville, CA, was determined by genetic (microsatellite) analyses. Twenty-one colonies of R. hesperus inhabited the six baited sites and eight colonies of R. hesperus occurred in the six unbaited sites. Five criteria provided a cause-and-effect link between the deployment of lufenuron termite bait and elimination of baited colonies: 1) association of foragers, as members of the same colony, in the independent monitoring stations and bait stations; 2) quantity of bait consumed; 3) abnormal physical appearance of foragers in bait stations; 4) disappearance of foragers from, and cessation of feeding in, independent monitoring stations visited by baited colonies; and 5) presence of foragers from, and continuation of feeding in, independent monitors visited by unbaited colonies. Baited colonies were devoid of foraging termites within a mean of 70.6 d (range, 37-93 d) of bait deployment. Colonies consumed a mean of 8.0 g of bait (range, 2.2-16.0 g). Wood consumption by baited and unbaited colonies was not significantly different during the 2 mo before baiting, 281.4 versus 590.5 mg/d per colony, respectively, nor during the 3 mo immediately after baiting, 112.5 versus 436.8 mg/d per colony, respectively. However, from 10 to 16 mo after baiting, wood consumption by baited colonies essentially ceased and was significantly less than the unbaited colonies, 7.9 versus 470.1 mg/d per colony, respectively.

Efficacy of verbenone for protecting ponderosa pine stands from western pine beetle (Coleoptera: Curculionidae: Scolytinae) attack in California.

The western pine beetle, Dendroctonus brevicomis LeConte (Coleoptera: Curculionidae: Scolytinae), is a major cause of ponderosa pine, Pinus ponderosa Dougl. ex Laws., mortality in much of western North America. Currently, techniques for managing D. brevicomis infestations are limited. Verbenone (4,6,6-trimethylbicyclo [3.1.1] hept-3-en-2-one) is an antiaggregation pheromone of several Dendroctonus spp., including D. brevicomis, and it has been registered as a biopesticide for control of mountain pine beetle, Dendroctonus ponderosae Hopkins, and southern pine beetle, Dendroctonus frontalis Zimmermann. We evaluated the efficacy of a 5-g verbenone pouch [82%-(-); 50 mg/d] applied at 125 Ulha for protecting P. ponderosa stands (2 ha) from D. brevicomis attack over a 3-yr period. No significant differences in levels of D. brevicomis-caused tree mortality or the percentage of unsuccessfully attacked trees were found between verbenone-treated and untreated plots during each year or cumulatively over the 3-yr period. Laboratory analyses of release rates and chemical composition of volatiles emanating from verbenone pouches after field exposure found no deterioration of the active ingredient or physical malfunction of the release device. The mean release rate of pouches from all locations and exposure periods was 44.5 mg/d. In a trapping bioassay, the range of inhibition of the 5-g verbenone pouch was determined to be statistically constant 2 m from the release device. We discuss the implications of these and other results to the development of verbenone as a semiochemical-based tool for management of D. brevicomis infestations in P. ponderosa stands.

Cuticular hydrocarbons and soldier defense secretions of Reticulitermes in southern California: a critical analysis of the taxonomy of the genus in North America.

Cuticular hydrocarbons (CHC) and soldier defense secretions (SDS) were characterized for collections of Reticulitermes from six counties (Los Angeles, Orange, Riverside, San Bernardino, San Diego, and Santa Barbara) in southern California. Collection sites included the type locality for R. hesperus, Lake Arrowhead (formerly known as Little Bear Lake) in the San Bernardino Mountains. In southern California, there are two CHC phenotypes, SC-A and SC-B, which are easily distinguished by the presence or absence of 5-methyl pentacosane, 5-methyl heptacosane, 5,17-dimethyl pentacosane, and 5,17-dimethyl heptacosane. These phenotypes are similar, but not identical, to previously designated phenotypes of Reticulitermes from northern California. The SDS of termites collected from southern California were characterized; (-)-germacrene A was abundant in all but the four samples from Lake Arrowhead. Soldiers of phenotype SC-A produced >79% germacrene A. The four samples from Lake Arrowhead produced no germacrene A, but contained >78% gamma-cadinene. The SDS from the Lake Arrowhead samples were more similar to those of CA-A/CA-A' from northern California than to any of the CHC phenotypes from southern California. Soldiers of CHC phenotype SC-B produced germacrene A, with the proportion varying from 16.2 to 98.7%. The SDS of SC-B were more similar to those of SC-A than to any of the phenotypes from northern California. The CHC phenotype SC-A found in southern California likely represents R. hesperus and SC-B appears to be a new, as yet undescribed species. We discuss the state of current taxonomic research on Reticulitermes.

Identification of termite species by the hydrocarbons in their feces.

Blends of abundant cuticular hydrocarbons are species-specific for termites (Isoptera) and can be used to identify a given taxon without the diagnostic castes, soldiers or adults. We demonstrate that hydrocarbon extracts of termite fecal pellets from damaged wood can also be characterized and used to identify termites responsible for damage, even though termites are no longer present or easily recovered. In structures infested by drywood termites, it is common to find fecal pellets, but difficult to extract termites from the finished wood in service. Nine species belonging to two families (Kalotermitidae and Termopsidae) were examined to compare the hydrocarbon composition of termites and their fecal pellets. Diversity was extensive: at least one half of the amount of the hydrocarbons from Neotermes connexus, Incisitermes immigrans, Cryptotermes brevis, Cryptotermes cynocephalus, Procryptotermes corniceps, and Zootermopsis nevadensis nuttingi was olefins. Incisitermes minor and Pterotermes occidentis incorporated only small amounts of olefins in cuticular hydrocarbons; Marginitermes hubbardi had no detectable olefins. Hydrocarbons extracted from fecal pellets were qualitatively and quantitatively similar to cuticular extracts and can be used to determine the termite species responsible without the termites present.

Phylogenetic analyses of mtDNA sequences corroborate taxonomic designations based on cuticular hydrocarbons in subterranean termites.

Cuticular hydrocarbons (CHCs) are valuable characters for the analysis of cryptic insect species with few discernible morphological characters. Yet, their use in insect systematics, specifically in subterranean termites in the genus Reticulitermes (Isoptera: Rhinotermitidae), remains controversial. In this paper, we show that taxonomic designations in Reticulitermes from California (USA) suggested in light of differences among CHC phenotypes are corroborated by phylogenetic analyses using mtDNA sequences. Analyses based on CHC phenotypes and supported, in part, by behavioral and ecological differences have suggested the presence of more species than the two currently recognized: R. hesperus Banks and R. tibialis Banks. We analyze a 680 base pair fragment of the mitochondrial DNA cytochrome oxidase (COII) gene from 45 new (21 collection localities) and two previously recorded samples of Reticulitermes from California using parsimony and maximum likelihood methods. Both methods result in trees with highly similar topologies. Bootstrapping indicates support for six clades of Reticulitermes, and corroborates groupings based on cuticular hydrocarbons. One of the clades, R. hesperus, is already recognized in California, while four clades appear to be previously undescribed taxa. Although identification of the final clade is inconclusive, it includes a sample putatively identified as R. tibialis. Therefore, using phylogenetic analyses we corroborate chemical characters used to identify taxa, associate a chemical phenotype with a previously described species, and provide additional support for undescribed taxa of Reticulitermes.

Cuticular hydrocarbons suggest three lineages in Reticulitermes (Isoptera: Rhinotermitidae) from North America.

Cuticular hydrocarbon mixtures can be used to discriminate insect taxa. They have utility for determining phylogenetic relationships where they are independent characters with discrete states and represent a hierarchical distribution of shared, derived characters. We report inferred degrees of relatedness among the chemical phenotypes of Reticulitermes from PAUP (phylogenetic analysis using parsimony) analyses of cuticular hydrocarbon characters. One hundred and forty-one Reticulitermes colonies collected from California, Georgia, New Mexico, Arizona and Nevada were used. Initial maximum parsimony analyses sorted the 141 colonies into 26 chemical phenotypes. Subsequent analyses, using the ancestral species Coptotermes formosanus and Heterotermes sp. as outgroups, sorted Reticulitermes taxa into three major lineages, each characterized by a different set of dominant methyl-branched or unsaturated hydrocarbon components. Reticulitermes in lineage I have cuticular hydrocarbon mixtures with a preponderance of internally branched monomethylalkanes and 11,15-dimethylalkanes. Those in lineage II are defined by a preponderance of 5-methylalkanes and 5,17-dimethylalkanes. Taxa in lineage III are characterized by the predominance of olefins and a relative paucity of n-alkanes and methyl-branched alkanes. Bootstrap analyses and decay indices provided statistical support and robustness for these chemical-based relationships.