Development of an array of molecular tools for the identification of khapra beetle (Trogoderma granarium), a destructive beetle of stored food products.

Trogoderma granarium Everts, the khapra beetle, native to the Indian subcontinent, is one of the world's most destructive pests of stored food products. Early detection of this pest facilitates prompt response towards the invasion and prevents the need for costly eradication efforts. Such detection requires proper identification of T. granarium, which morphologically resembles some more frequently encountered, non-quarantine congeners. All life stages of these species are difficult to distinguish using morphological characters. Additionally, biosurveillance trapping can result in the capture of large numbers of specimens awaiting identification. To address these issues, we aim to develop an array of molecular tools to rapidly and accurately identify T. granarium among non-target species. Our crude, cheap DNA extraction method performed well for Trogoderma spp. and is suitable for downstream analyses including sequencing and real-time PCR (qPCR). We developed a simple quick assay usingrestriction fragment length polymorphism to distinguish between T. granarium and the closely related, congeneric T. variabile Ballion and T. inclusum LeConte. Based on newly generated and published mitochondrial sequence data, we developed a new multiplex TaqMan qPCR assay for T. granarium with improved efficiency and sensitivity over existing qPCR assays. These new tools benefit regulatory agencies and the stored food products industry by providing cost- and time-effective solutions to enhance the identification of T. granarium from related species. They can be added to the existing pest detection toolbox. The selection of which method to use would depend on the intended application.

Direct competition and potential displacement involving managed Trogoderma stored product pests.

The establishment of an exotic pest may require displacing local species with a similar niche. The potential of Trogoderma granarium to displace Trogoderma inclusum was explored in a stored product setting. We performed direct competition experiments varying commodity and temperature over different durations. At nine weeks T. inclusum outproduced T. granarium on all commodities at any temperature. However the proportion of T. granarium versus T. inclusum was greater at 32 °C compared to 25 °C. The nine-week production of T. granarium was best on wheat, while rice was optimal for T. inclusum. After 25 weeks, when adults were used at the start of competition, T. inclusum maintained an advantage in the direct competition. If larvae were used to initiate the competition for 25 weeks, the two species coexisted well at 25 °C, but T. granarium nearly excluded T. inclusum at 32 °C. Thus T. inclusum performs better in competition over shorter intervals when resources are plentiful, but T. granarium can be more successful over longer time periods, particularly when late instar larvae are involved. The finding suggests a real threat of introductions of T. granarium larvae to establish populations within grain storage infrastructure where T. inclusum is common.

Cryptic genetic diversity and associated ecological differences of Anastatus orientalis, an egg parasitoid of the spotted lanternfly.

Anastatus orientalis, native to northern China, is an egg parasitoid wasp of the spotted lanternfly (Lycorma delicatula) and is being tested as a potential biological control agent for invasive L. delicatula in the United States. As a component of these evaluations, live A. orientalis collected from Beijing and Yantai in China were reared in containment in the U.S. These specimens showed different responses in diapause behaviors to rearing conditions used previously by other researchers. To understand the primary mechanism potentially driving discrepancies in important life history traits, we used molecular tools to examine the genetic composition of A. orientalis from China and from South Korea, where the parasitoid has been introduced to aid in the population management of invasive L. delicatula. Molecular analysis of mitochondrial DNA recovered six haplotype groups, which exhibit biased frequency of abundance between collection sites. Some haplotypes are widespread, and others only occur in certain locations. No apparent pattern is observed between wasps collected from different years or emergence seasons. Uncorrected genetic distances between haplotype groups range from 0.44% to 1.44% after controlling for within-group variation. Genetic variance of A. orientalis is characterized by high levels of local diversity that contrasts with a lack of a broad-scale population structure. The introduced Korean population exhibits lower genetic diversity compared to native populations. Additionally, we created iso-female lines for major haplotype groups through laboratory rearing. Differences in diapause behavior were correlated with mitochondrial haplotype. Our results indicate that the observed life history traits in A. orientalis have a genetic base.

Tracking invasions of a destructive defoliator, the gypsy moth (Erebidae: Lymantria dispar): Population structure, origin of intercepted specimens, and Asian introgression into North America.

Genetic data can help elucidate the dynamics of biological invasions, which are fueled by the constant expansion of international trade. The introduction of European gypsy moth (Lymantria dispar dispar) into North America is a classic example of human-aided invasion that has caused tremendous damage to North American temperate forests. Recently, the even more destructive Asian gypsy moth (mainly L. d. asiatica and L. d. japonica) has been intercepted in North America, mostly transported by cargo ships. To track invasion pathways, we developed a diagnostic panel of 60 DNA loci (55 nuclear and 5 mitochondrial) to characterize worldwide genetic differentiation within L. dispar and its sister species L. umbrosa. Hierarchical analyses supported strong differentiation and recovered five geographic groups that correspond to (1) North America, (2) Europe plus North Africa and Middle East, (3) the Urals, Central Asia, and Russian Siberia, (4) continental East Asia, and (5) the Japanese islands. Interestingly, L. umbrosa was grouped with L. d. japonica, and the introduced North American population exhibits remarkable distinctiveness from contemporary European counterparts. Each geographic group, except for North America, shows additional lower-level structures when analyzed individually, which provided the basis for inference of the origin of invasive specimens. Two assignment approaches consistently identified a coastal area of continental East Asia as the major source for Asian invasion during 2014-2015, with Japan being another source. By analyzing simulation and laboratory crosses, we further provided evidence for the occurrence of natural Asian-North American hybrids in the Pacific Northwest, raising concerns for introgression of Asian alleles that may accelerate range expansion of gypsy moth in North America. Our study demonstrates how genetic data contribute to bio-surveillance of invasive species with results that can inform regulatory management and reduce the frequency of trade-associated invasions.