Conifer bark beetles.

Fettig and Audley introduce the bark beetles-a large and diverse group of insects of which some are commonly recognized as important disturbance agents in conifer forests.

Cross-scale interaction of host tree size and climatic water deficit governs bark beetle-induced tree mortality.

The recent Californian hot drought (2012-2016) precipitated unprecedented ponderosa pine (Pinus ponderosa) mortality, largely attributable to the western pine beetle (Dendroctonus brevicomis; WPB). Broad-scale climate conditions can directly shape tree mortality patterns, but mortality rates respond non-linearly to climate when local-scale forest characteristics influence the behavior of tree-killing bark beetles (e.g., WPB). To test for these cross-scale interactions, we conduct aerial drone surveys at 32 sites along a gradient of climatic water deficit (CWD) spanning 350 km of latitude and 1000 m of elevation in WPB-impacted Sierra Nevada forests. We map, measure, and classify over 450,000 trees within 9 km2, validating measurements with coincident field plots. We find greater size, proportion, and density of ponderosa pine (the WPB host) increase host mortality rates, as does greater CWD. Critically, we find a CWD/host size interaction such that larger trees amplify host mortality rates in hot/dry sites. Management strategies for climate change adaptation should consider how bark beetle disturbances can depend on cross-scale interactions, which challenge our ability to predict and understand patterns of tree mortality.

A one-dimensional map to study multi-seasonal coffee infestation by the coffee berry borer.

The coffee berry borer (CBB, Hypothenemus hampei) is the most serious insect pest of coffee worldwide; understanding the dynamics of its reproduction is essential for pest management. The female CBB penetrates the coffee berry, eats the seed, and reproduces inside it. A mathematical model of the infestation progress of the coffee berry by the CBB during several coffee seasons is formulated. The model represents the interaction among five populations: uninfested, slightly infested, and severely infested coffee berries, and free and encapsulated CBBs. Coffee harvesting is also included in the model. A one-dimensional map is derived for tracking the population dynamics subject to certain coffee harvesting percentages over several seasons. Stability analysis of the map's fixed points shows that CBB infestation could be eliminated or controlled to a specific level over multiple seasons of coffee harvesting. However, the percent of coffee harvesting required is determined by the level of CBB infestation at the beginning of the first season and in some cases it is impossible to achieve that percentage.

Storage of corn seeds infested by weevil

Weevil is one of the main pests of stored corn. The adults feed on intact and broken seeds or bran, while the larvae develop by feeding on the inside of the seeds, resulting in a reduction in weight and physiological quality. The objective of this study was to evaluate the effects of Sitophilus zeamais infestation on corn seeds during storage. Corn seeds were infested using insect densities: 0, 36, 72, 108, and 144 individuals per 240 g of seeds. The infested seeds were placed in individualized plastic containers and covered with voile fabric. The seeds were stored for 60 days under laboratory conditions with an average temperature of around 27 °C and relative humidity of 85%. Initially and every 15 days of storage for 60 days, the seeds were evaluated using the parameters: number of S. zeamais individuals, total number of infested seeds, seed weight loss, germination test and seedling emergence in the tray. The population increase of S. zeamais causes a decrease on the physical and physiological quality of stored corn seeds, but the deterioration rate does not increase in the same proportion as the weevil population growth rate.

Seasonal changes in population structure of the ambrosia beetle Xylosandrus compactus and its associated fungi in a southern Mediterranean environment.

Exotic ambrosia beetles are increasing in Europe due to global trade and global warming. Among these xylomycetophagous insects, Xylosandrus compactus (Eichhoff) (Coleoptera: Curculionidae) is a serious threat for several Mediterranean host plants. Carob trees growing in Sicily (Italy) have been extensively attacked by beetles leading to rapid tree decline. Although X. compactus has been found in Europe for several years, most aspects of its ecology are still unknown. We thus studied the population structure and dynamics of X. compactus, together with its twig size preference during a sampling of infested carob trees in south east Sicily. In addition, fungi associated with insects or galleries were isolated and characterized. The results showed that, in this newly-colonized environment and host plant, adult X. compactus overwinters inside twigs and starts to fly and reproduce in mid spring, completing five generations before overwintering in late fall. The mean diameter of carob twigs infested by the beetle varied significantly over the seasons, with the insect tending to infest larger twigs as season progresses. The mean number of adults/gallery was 19.21, ranging from 6 to 28. The minimum temperature significantly affected the overwintering adult mortality. Ambrosiella xylebori and Fusarium solani were the main symbionts associated with the pest in this study. Acremonium sp. was instead recorded for the first time in Europe inside X. compactus galleries. Several other fungi species were also found for the first time in association with X. compactus. Our findings provide useful insights into the sustainable management of this noxious pest.

Genetic Basis of Maize Resistance to Multiple Insect Pests: Integrated Genome-Wide Comparative Mapping and Candidate Gene Prioritization.

Several species of herbivores feed on maize in field and storage setups, making the development of multiple insect resistance a critical breeding target. In this study, an association mapping panel of 341 tropical maize lines was evaluated in three field environments for resistance to fall armyworm (FAW), whilst bulked grains were subjected to a maize weevil (MW) bioassay and genotyped with Diversity Array Technology's single nucleotide polymorphisms (SNPs) markers. A multi-locus genome-wide association study (GWAS) revealed 62 quantitative trait nucleotides (QTNs) associated with FAW and MW resistance traits on all 10 maize chromosomes, of which, 47 and 31 were discovered at stringent Bonferroni genome-wide significance levels of 0.05 and 0.01, respectively, and located within or close to multiple insect resistance genomic regions (MIRGRs) concerning FAW, SB, and MW. Sixteen QTNs influenced multiple traits, of which, six were associated with resistance to both FAW and MW, suggesting a pleiotropic genetic control. Functional prioritization of candidate genes (CGs) located within 10-30 kb of the QTNs revealed 64 putative GWAS-based CGs (GbCGs) showing evidence of involvement in plant defense mechanisms. Only one GbCG was associated with each of the five of the six combined resistance QTNs, thus reinforcing the pleiotropy hypothesis. In addition, through in silico co-functional network inferences, an additional 107 network-based CGs (NbCGs), biologically connected to the 64 GbCGs, and differentially expressed under biotic or abiotic stress, were revealed within MIRGRs. The provided multiple insect resistance physical map should contribute to the development of combined insect resistance in maize.

Organic mulches reduce crop attack by sweetpotato weevil (Cylas formicarius).

Mulching with organic materials is a management practice with long history for weed suppression, soil water conservation and erosion control. Its potential impact on crop pests is less well explored. Here we report its utility for reducing crop damage by the serious pest, sweetpotato weevil (Cylas formicarius). Laboratory bioassays measured the response of adult female weevils to sweetpotato storage roots beneath mulches of fresh or dried plant materials. Weevils were significant repelled by fresh basil, catnip, basil lime and dry eucalyptus, cypress, lucerne and sugarcane. A subsequent field study found that mulches of dry cypress, eucalyptus and lucerne reduced movement of weevils from a release point to reach sweetpotato plants and lowered level of damage to storage roots. Results demonstrate that mulching with organic materials merits further testing as part of the integrated management of sweetpotato weevil, particularly to protect developing storage roots during dry periods when soil cracking can facilitate access by pests.

Construction of a high density linkage map and genome dissection of bruchid resistance in zombi pea (Vigna vexillata (L.) A. Rich).

Zombi pea (Vigna vexillata) is a legume crop that is resistant to several biotic and abiotic stresses. Callosobruchus maculatus and Callosobruchus chinensis are serious stored-insect pests of legume crops. We constructed a high-density linkage map and performed quantitative trait loci (QTLs) mapping for resistance to these insect species in zombi pea. An F2 population of 198 individuals from a cross between 'TVNu 240' (resistant) and 'TVNu 1623' (susceptible) varieties was used to construct a linkage map of 6,529 single nucleotide polymorphism markers generated from sequencing amplified fragments of specific loci. The map comprised 11 linkage groups, spanning 1,740.9 cM, with an average of 593.5 markers per linkage group and an average distance of 0.27 cM between markers. High levels of micro-synteny between V. vexillata and cowpea (Vigna unguiculata), mungbean (Vigna radiata), azuki bean (Vigna angularis) and common bean (Phaseolus vulgaris) were found. One major and three minor QTLs for C. chinensis resistance and one major and one minor QTLs for C. maculatus resistance were identified. The major QTLs for resistance to C. chinensis and C. maculatus appeared to be the same locus. The linkage map developed in this study will facilitate the identification of useful genes/QTLs in zombi pea.

Development of an SNP-based high-density linkage map and QTL analysis for bruchid (Callosobruchus maculatus F.) resistance in black gram (Vigna mungo (L.) Hepper).

Black gram (Vigna mungo var. mungo) is an important pulse crop in Asia. The cowpea weevil (Callosobruchus maculatus) is a stored-seed insect pest (seed weevil/bruchid) that causes serious postharvest losses in pulse crops, including black gram. In this study, we constructed a high-density linkage map for black gram and identified quantitative trait loci (QTLs) for C. maculatus resistance. A recombinant inbred line (RIL) population of 150 lines from a cross between BC48 [cultivated black gram (var. mungo); bruchid-susceptible] and TC2210 [wild black gram (var. silvestris); bruchid-resistant] were used to construct a linkage map of 3,675 SNP markers from specific-locus amplified fragment sequencing. The map comprised 11 linkage groups spanning 1,588.7 cM with an average distance between adjacent markers of 0.57 cM. Seeds of the RIL population grown in 2016 and 2017 were evaluated for C. maculatus resistance through two traits; the percentage of damaged seeds (PDS) and infestation severity progress (AUDPS). Inclusive composite interval mapping identified three QTLs each for PDS and AUDPS. Two QTLs, qVmunBr6.1 and qVmunBr6.2, mapped about 10 cM apart on linkage group 6 were common between PDS and AUDPS. Comparative genome analysis revealed that qVmunBr6.1 and qVmunBr6.2 are new loci for C. maculatus resistance in Vigna species and that genes encoding a lectin receptor kinase and chitinase are candidates for qVmunBr6.2. The high-density linkage map constructed and QTLs for bruchid resistance identified in this study will be useful for molecular breeding of black gram.

Overexpression of the ZmDEF1 gene increases the resistance to weevil larvae in transgenic maize seeds.

Plant defensins are divided into 18 groups and are multifunctional proteins. The Zea mays defensin 1 (ZmDEF1) gene encodes the defensin 1 protein, which can inhibit alpha-amylase in the insect gut. In this study, the ZmDEF1 gene was transferred into two maize cultivars, LC1 and LVN99, to improve weevil resistance in maize. The recombinant ZmDEF1 protein was assessed for its ability to inhibit alpha-amylase in the gut of the larvae of the maize weevil (Sitophilus zeamais Motsch.). ZmDEF1 was cloned into a pBetaPhaso-dest vector, which harbours phaseolin, a seed-specific promoter, and the Agrobacterium tumefaciens strain C58 harbouring the pBetaPhaso-ZmDEF1 vector was used to transfer the ZmDEF1 gene into two maize cultivars using immature embryos. Transformed calluses were selected on selection media containing kanamycin. The stable integration of the ZmDEF1 transgene into the transgenic maize plant genome was confirmed using Southern blotting. The recombinant ZmDEF1 protein of approximately 10 kDa was expressed in three transgenic maize lines from the LC1 cultivar (C1, C3, and C5) and two transgenic maize lines from the LVN99 cultivar (L1 and L3). The ZmDEF1 transgenic efficiency based on the results of PCR, as well as Southern and Western blotting, was 1.32% and 0.82%, respectively, which depends on the genotypes of LC1 and LVN99. The recombinant ZmDEF1 protein inhibited the alpha-amylase activity of the maize weevil larvae, and its ability to inhibit alpha-amylase is 54.52-63.09% greater than the ZmDEF1 protein extracted from non-transgenic plants.

Fine-tuned intruder discrimination favors ant parasitoidism.

A diversity of arthropods (myrmecophiles) thrives within ant nests, many of them unmolested though some, such as the specialized Eucharitidae parasitoids, may cause direct damage to their hosts. Ants are known to discriminate between nestmates and non-nestmates, but whether they recognize the strength of a threat and their capacity to adjust their behavior accordingly have not been fully explored. We aimed to determine whether Ectatomma tuberculatum ants exhibited specific behavioral responses to potential or actual intruders posing different threats to the host colony and to contribute to an understanding of complex ant-eucharitid interactions. Behavioral responses differed significantly according to intruder type. Ants evicted intruders that represented a threat to the colony's health (dead ants) or were not suitable as prey items (filter paper, eucharitid parasitoid wasps, non myrmecophilous adult weevils), but killed potential prey (weevil larvae, termites). The timing of detection was in accordance with the nature and size of the intruder: corpses (a potential source of contamination) were detected faster than any other intruder and transported to the refuse piles within 15 min. The structure and complexity of behavioral sequences differed among those intruders that were discarded. Workers not only recognized and discriminated between several distinct intruders but also adjusted their behavior to the type of intruder encountered. Our results confirm the previously documented recognition capabilities of E. tuberculatum workers and reveal a very fine-tuned intruder discrimination response. Colony-level prophylactic and hygienic behavioral responses through effective removal of inedible intruders appears to be the most general and flexible form of defense in ants against a diverse array of intruders. However, this generalized response to both potentially lethal and harmless intruders might have driven the evolution of ant-eucharitid interactions, opening a window for parasitoid attack and allowing adult parasitoid wasps to quickly leave the natal nest unharmed.

Genetic modification of asexual Epichloë endophytes with the perA gene for peramine biosynthesis.

Development of grass-endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.

Identification and expression profiles of twenty-six glutathione S-transferase genes from rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae).

The rice weevil, Sitophilus oryzae, is one of the most destructive pests in stored cereals products. In this study, 26 cDNAs encoding glutathione S-transferases (GSTs) were sequenced and characterized in S. oryzae. Phylogenetic analysis displayed the categorization of 26 GSTs into six different cytosolic classes, including two in the delta, twelve in epsilon, three in omega, six in sigma, two in theta, and one in zeta class. RT-qPCR assay illustrated that the relative expression of ten GST genes was significantly higher in adult stages than in larval and pupal developmental stages. Tissue-specific expression analysis revealed that the SoGSTe5, SoGSTe7, SoGSTe12, and SoGSTz1 were up-regulated in the midgut, SoGSTe2, SoGSTe6, and SoGSTs2 were up-regulated in the fat body, and three GSTs (SoGSTd1, SoGSTd2 and SoGSTe4) were up-regulated in Malpighian tubules. RT-qPCR indicated that five GST genes were over expressed after exposure to phosphine at various times and concentrations. The increase in GST gene expressions after phosphine exposure in S. oryzae may lead to an improved tolerance for fumigations and xenobiotics.

Spatial distribution and losses by grain destroying insects in transgenic corn expressing the toxin Cry1Ab.

Insect pests are one of the factors that most impact plant yield. The magnitude of the losses and the spatiotemporal pest distribution in crops is a result of their interactions with the environment. Therefore, the understanding of the causes of production losses and the pest spatial patterns is important for the development of suitable sampling plans and pest management programs. Thus, this study aimed to quantify grain losses caused by insects and to determine the spatial distribution pattern of arthropod pest species in Bt and non-Bt corn. The prevailing insect pests in the corn ears were the earworm and fall armyworm caterpillars (Helicoverpa spp. and Spodoptera frugiperda), the cornsilk fly (Euxesta spp.), the maize weevil (Sitophilus zeamais), and the square-necked grain beetle (Cathartus quadricollis). The non-Bt corn was more attacked by the caterpillars and the weevil, while Bt corn was more affected by the cornsilk fly Euxesta spp. Spatial dependence was significant for the damage caused by the caterpillars, the grain beetle and the maize weevil in both the Bt and non-Bt corn genotypes. The range of the damage caused by the insects was between 9.0-9.7 m for the caterpillars, 6.9-12.20 m for the cornsilk fly, 10.7-80.4 m for the square-necked grain beetle, and 51.9-170.7 m for the maize weevil. The pattern of the spatial distribution of pest damage in both corn genotypes (i.e., Bt and non-Bt corn) was similar with a prevalence of moderate to strong spatial dependence and aggregate damage distribution. The plants near to the sampling points exhibited injury and infestation levels similar to those of the sampled plants.

Evaluation of nine genotypes of oilseed rape (Brassica napus L.) for larval infestation and performance of rape stem weevil (Ceutorhynchus napi Gyll.).

The rape stem weevil, Ceutorhynchus napi Gyll., is a serious pest of winter oilseed rape (Brassica napus L.) crops in Europe causing severe yield loss. In currently used oilseed rape cultivars no resistance to C. napi has been identified. Resynthesized lines of B. napus have potential to broaden the genetic variability and may improve resistance to insect pests. In this study, the susceptibility to C. napi of three cultivars, one breeding line and five resynthesized lines of oilseed rape was compared in a semi-field plot experiment under multi-choice conditions. Plant acceptance for oviposition was estimated by counting the number of C. napi larvae in stems. The larval instar index and the dry body mass were assessed as indicators of larval performance. The extent of larval feeding within stems was determined by the stem injury coefficient. Morphological stem traits and stem contents of glucosinolates were assessed as potential mediators of resistance. The resynthesized line S30 had significantly fewer larvae than the cultivars Express617 and Visby and the resynthesized lines L122 and L16. The low level of larval infestation in S30 was associated with a low larval instar and stem injury index. Low numbers of larvae were not correlated with the length or diameter of stems, and the level of stem glucosinolates. As indicated by the low larval infestation and slow larval development the resistance of S30 to C. napi is based on both antixenotic and antibiotic properties of the genotypes. The resynthesized line S30 should therefore be introduced into B. napus breeding programs to enhance resistance against C. napi.

Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2-ΔΔCt analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 µg g-1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 µg g-1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.

Pre-dispersal strategies by Quercus schottkyana to mitigate the effects of weevil infestation of acorns.

We investigated how pre-dispersal strategies may mitigate the effects of weevil infestation of acorns in a population of Quercus schottkyana, a dominant oak in Asian evergreen broad-leaved forests, and assess if weevil infestation contributes to low seedling recruitment. We counted the number of acorns produced, daily from the end of August to mid-late November for 9 years from 2006-2014. We also recorded the rate of acorn infestation by weevils and acorn germination rates of weekly collections. Annual acorn production was variable, but particularly low in 2011 and 2013. There was no trade-off between acorn production and acorn dry mass. However, acorns produced later in the season were significantly heavier. For most years: (i) the rate of weevil infestation was negatively density dependent (a greater proportion of acorns died with increased acorn density), (ii) the percentage germination of acorns was positively density dependent (proportionately more acorns germinated with increased density), and (iii) as the season progressed, the percentage of infested acorns declined while germination rates increased. Finally, (iv) maximum acorn production, percentage infestation and percentage germination were asynchronous. Although pre-dispersal mortality is important it is unlikely to be the primary factor leading to low recruitment of oak seedlings.

Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants.

Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular evolution to the cry8Ka1 gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC50 2.83 µg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods.

A Model for Mountain Pine Beetle Outbreaks in an Age-Structured Forest: Predicting Severity and Outbreak-Recovery Cycle Period.

The mountain pine beetle (MPB, Dendroctonus ponderosae), a tree-killing bark beetle, has historically been part of the normal disturbance regime in lodgepole pine (Pinus contorta) forests. In recent years, warm winters and summers have allowed MPB populations to achieve synchronous emergence and successful attacks, resulting in widespread population outbreaks and resultant tree mortality across western North America. We develop an age-structured forest demographic model that incorporates temperature-dependent MPB infestations. Stability of fixed points is analyzed as a function of (thermally controlled) MPB population growth rates and indicates the existence of periodic outbreaks that intensify as growth rates increase. We devise analytical methods to predict outbreak severity and duration as well as outbreak return time. After incorporating a spatial aspect and controlling initial stand demographic variation, the model predicts cycle periods that fall within observed outbreak return time ranges. To assess future MPB impact on forests, we use climate model projected temperatures with our model-based approximation methods to predict potential severity of future outbreaks that reflect the effects of changing climate.

Plasticity and evolution of (+)-3-carene synthase and (-)-sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance.

The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (-)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3-carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (-)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene, whereas phenylalanine promotes (-)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects.