Early evidence of establishment of the tropical bedbug (Cimex hemipterus) in Central Europe.

In recent decades, the world has witnessed a remarkable resurgence of bedbugs (Hemiptera: Cimicidae). Although populations of the common bedbug, Cimex lectularius L., expanded in temperate regions of its original distribution, the tropical bedbug, C. hemipterus (F.), increased its abundance in warmer regions, where it also had been historically distributed. However, C. hemipterus has recently been observed to be expanding to other areas, e.g. North Australia, Middle East, the United States and Russia. In other parts of Europe, few sporadic and ephemeral introductions of C. hemipterus were recorded until recently. We conducted an extensive sampling of European bedbug populations starting in 2002 and found that C. hemipterus has recently become locally established. Among 566 examined infestations, nearly all of which involved C. lectularius, C. hemipterus occurred in six infestations collected since 2019. In at least three cases, the social background of inhabitants of the infested properties indicated that tropical bedbugs likely spread within local communities. Using cytochrome oxidase subunit I, we linked five of the infestations to the most common haplotype found globally, and one to an African haplotype. In all infestations, we observed two kdr-associated mutations in the sodium channel gene, which are also commonly found across the world.

Identification of Reticulitermes Subterranean Termites (Blattodea: Rhinotermitidae) in the Eastern United States Using Inter-Simple Sequence Repeats.

In the eastern United States, there are nine species of subterranean termites in three genera: Reticulitermes (six species), Coptotermes (two species), and Prorhinotermes (one species). These species serve as important ecological players by decomposing cellulose material, and some are important structural pests. Many of these species are difficult to discriminate morphologically and require examining the reproductive or soldier castes, which can be difficult to collect. While some genetic tools have been developed for species identification, they are often expensive and time-consuming. To help facilitate identification, we developed a more cost-effective and rapid genetic method to identify Reticulitermes species by screening 10 PCR primers that amplified inter-simple sequence repeats (ISSRs) in other termite species. From these, one primer was amplified in all five focal Reticulitermes species and contained conserved, species-specific fragments. We further screened this identification method on samples of each species covering a diversity of mitochondrial DNA haplotypes and localities. This identification method utilizing ISSRs can be used to quickly identify five species of Reticulitermes subterranean termites in the eastern United States in a matter of hours, providing a useful technique for pest management as well as future ecological research.

Development of a universal double-digest RAD sequencing approach for a group of nonmodel, ecologically and economically important insect and fish taxa.

The generation of genome-scale data is critical for a wide range of questions in basic biology using model organisms, but also in questions of applied biology in nonmodel organisms (agriculture, natural resources, conservation and public health biology). Using a genome-scale approach on a diverse group of nonmodel organisms and with the goal of lowering costs of the method, we modified a multiplexed, high-throughput genomic scan technique utilizing two restriction enzymes. We analysed several pairs of restriction enzymes and completed double-digestion RAD sequencing libraries for nine different species and five genera of insects and fish. We found one particular enzyme pair produced consistently higher number of sequence-able fragments across all nine species. Building libraries off this enzyme pair, we found a range of usable SNPs between 4000 and 37 000 SNPS per species and we found a greater number of usable SNPs using reference genomes than de novo pipelines in STACKS. We also found fewer reads in the Read 2 fragments from the paired-end Illumina Hiseq run. Overall, the results of this study provide empirical evidence of the utility of this method for producing consistent data for diverse nonmodel species and suggest specific considerations for sequencing analysis strategies.

Extensive Mitochondrial Heteroplasmy in Natural Populations of a Resurging Human Pest, the Bed Bug (Hemiptera: Cimicidae).

Homoplasmy, the occurrence of a single mitochondrial DNA haplotype within an individual, has been the accepted condition across most organisms in the animal kingdom. In recent years, a number of exceptions to this rule have been reported, largely due to the ease with which single nucleotide polymorphisms can be detected. Evidence of heteroplasmy-two or more mitochondrial variants within a single individual-has now been documented in a number of invertebrates; however, when present, heteroplasmy usually occurs at low frequencies both within individuals and within populations. The implications of heteroplasmy may be far reaching, both to the individual in relation to its health and fitness, and when considering the evolutionary dynamics of populations. We present novel evidence for frequent mtDNA heteroplasmy in the bed bug, Cimex lectularius L. (Hemiptera: Cimicidae). Our findings show that heteroplasmy is common, with 5 of 29 (17%) populations screened exhibiting two mitochondrial variants in a ∼1:2 ratio within each individual. We hypothesize that the mechanism underlying heteroplasmy in bed bugs is paternal leakage because some haplotypes were shared among unrelated populations and no evidence for nuclear mitochondrial DNA sequences was detected.

Relationship between invasion success and colony breeding structure in a subterranean termite.

Factors promoting the establishment and colonization success of introduced populations in new environments constitute an important issue in biological invasions. In this context, the respective role of pre-adaptation and evolutionary changes during the invasion process is a key question that requires particular attention. This study compared the colony breeding structure (i.e. number and relatedness among reproductives within colonies) in native and introduced populations of the subterranean pest termite, Reticulitermes flavipes. We generated and analysed a data set of both microsatellite and mtDNA loci on termite samples collected in three introduced populations, one in France and two in Chile, and in the putative source population of French and Chilean infestations that has recently been identified in New Orleans, LA. We also provided a synthesis combining our results with those of previous studies to obtain a global picture of the variation in breeding structure in this species. Whereas most native US populations are mainly composed of colonies headed by monogamous pairs of primary reproductives, all introduced populations exhibit a particular colony breeding structure that is characterized by hundreds of inbreeding reproductives (neotenics) and by a propensity of colonies to fuse, a pattern shared uniquely with the population of New Orleans. These characteristics are comparable to those of many invasive ants and are discussed to play an important role during the invasion process. Our finding that the New Orleans population exhibits the same breeding structure as its related introduced populations suggests that this native population is pre-adapted to invade new ranges.

Genome size and ploidy of Thysanoptera.

Flow cytometry was used to study the genome sizes and ploidy levels for four thrips species: Franklinothrips orizabensis Johansen (Thysanoptera: Aeolothripidae), Frankliniella occidentalis Pergande, Frankliniella fusca Hinds, and Thrips tabaci Lindeman (Thysanoptera: Thripidae). F. orizabensis males and females had 1C genome sizes of 426 Mb and 422 Mb, respectively. Male and female F. fusca had 1C genome sizes of 392 Mb and 409 Mb, whereas F. occidentalis males and females had smaller 1C genomes that were 345 Mb and 337 Mb, respectively. Male F. orizabensis, F. occidentalis and F. fusca were haploid and females diploid. Five isofemale lines of T. tabaci, initiated from parthenogenetic, thelytokous females and collected from different locations in North Carolina, were included in this study; no males were available. One isofemale line was diploid with a genome size of 1C = 310 Mb, and the other four had a mean genome size of 1C = 482 Mb, which is consistent with evidence from microsatellite data of diploidy and polyploidy, respectively, in these same five thelytokous lines. This is the first study to produce genome size estimates for thysanopteran species, and report polyploidy in T. tabaci populations.

Global genetic analysis reveals the putative native source of the invasive termite, Reticulitermes flavipes, in France.

Biological invasions are recognized as a major threat to both natural and managed ecosystems. Phylogeographic and population genetic analyses can provide information about the geographical origins and patterns of introduction and explain the causes and mechanisms by which introduced species have become successful invaders. Reticulitermes flavipes is a North American subterranean termite that has been introduced into several areas, including France where introduced populations have become invasive. To identify likely source populations in the USA and to compare the genetic diversity of both native and introduced populations, an extensive molecular genetic study was undertaken using the COII region of mtDNA and 15 microsatellite loci. Our results showed that native northern US populations appeared well differentiated from those of the southern part of the US range. Phylogenetic analysis of both mitochondrial and nuclear markers showed that French populations probably originated from southeastern US populations, and more specifically from Louisiana. All of the mtDNA haplotypes shared between the United States and France were found in Louisiana. Compared to native populations in Louisiana, French populations show lower genetic diversity at both mtDNA and microsatellite markers. These findings are discussed along with the invasion routes of R. flavipes as well as the possible mechanisms by which French populations have evolved after their introduction.

Elimination of subterranean termite (Isoptera: Rhinotermitidae) colonies using a refined cellulose bait matrix containing noviflumuron when monitored and replenished quarterly.

Using a quarterly (3-mo) monitoring and bait-replenishment interval, 122 subterranean termite colonies throughout the United States were baited with a refined cellulose bait matrix containing 0.5% noviflumuron. All colonies were eliminated in less than 1 yr after initiation of baiting as determined by long-term monitoring and genetic markers. Sixty-three percent of the colonies were eliminated during the first quarter after the initiation of baiting and 77% of colonies were eliminated after consuming two bait tubes or less. This suggests that a single baiting cycle and bait installed in response to a single active monitoring device were sufficient to eliminate the majority of colonies. Although termites temporarily abandoned stations after depleting bait, workers resumed feeding when baits were replenished. Colonies that consumed large amounts of bait before elimination foraged into multiple stations, thus allowing adequate amounts of bait to sustain feeding. The time to eliminate termite colonies with bait replenished quarterly was similar to that previously reported for laminated cellulose bait replenished monthly. Our data support the conclusion that extending the bait replenishment interval from monthly to quarterly for bait tubes with refined cellulose containing 0.5% noviflumuron did not adversely impact colony elimination.

Identification and characterization of 15 polymorphic microsatellite loci in the western drywood termite, Incisitermes minor (Hagen).

Despite recognition of the western drywood termite, Incisitermes minor (Hagen), as one of the most economically important and destructive termite species in the USA, both its population and colony breeding structure genetically remain unclear. Here, we present primer sequences and initial characterization for 15 polymorphic microsatellite loci. In a sample of 30 individuals, representing six geographically distinct locations collected in California, USA, three to 15 alleles were detected segregating per locus. Within a single population observed heterozygosity ranged from 0.050 to 0.866.

Intraspecific variation and population structure of the German cockroach, Blattella germanica, revealed with RFLP analysis of the non-transcribed spacer region of ribosomal DNA.

Little information is available on genetic variation within and between populations of pest cockroaches. In this study, intraspecific HindIII polymorphism was investigated in the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera, Blattaria: Blattellidae), using restriction fragment length polymorphisms (RFLP) of the non-transcribed spacer (NTS) region of ribosomal DNA (rDNA). Individual male insects were collected from infestations at three different pig farms. Each population was characterized by HindIII restriction fragment frequencies and haplotype (a particular X-chromosome pattern) frequencies. The inheritance of the X-chromosome HindIII rDNA patterns over 12 generations (3 years) follows Mendelian patterns, and the stability of this polymorphic marker indicates infrequent genetic recombination of variable sites. Although pairwise genetic distance measures were uncorrelated with geographical distance, the pattern of genetic differentiation of the three cockroach populations suggests that human-mediated transport of cockroaches is an important force in shaping the population genetic structure of cockroach infestations, at least at the regional scale of 10-100 km. Sequence variation in the ribosomal NTS is a useful marker, and RFLP of rDNA is a simple, robust and reproducible technique for differentiating recently diverged cockroach populations.

The breeding system and population structure of the termite Reticulitermes grassei in Southwestern France.

We assessed colony and population structure in three French populations of the termite Reticulitermes grassei using eight polymorphic microsatellite loci. Although most colonies contained the offspring of multiple, highly related replacement reproductives (complex families), some contained the offspring of a single pair of reproductives (simple families), and the proportion of such colonies varied across populations. Populations also showed variability in the numbers of reproductives within complex families; the F-statistics of these families in one population were consistent with having upwards of 100 replacement reproductives, while in another population these colonies contained fewer than 10 pairs of reproductives. Colony boundaries in all populations were well defined, in spite of reports of a widespread breakdown in nestmate recognition and unicolonial populations of R. grassei from these regions in France. A second unexpected finding was a lack of significant isolation by distance among colonies within populations, indicating that colony reproduction by budding was rare or absent. The lack of this form of colony reproduction even within populations where it is expected to be common suggested that the propensity for colony budding may not be as common as suggested by the literature.

Queen control of sex ratio in fire ants.

The haplodiploid sex-determination system of ants gives rise to conflict between queens and workers over colony sex ratios, and the female-biased allocation ratios seen in many species suggest that workers often prevail in this conflict. We exchanged queens between male- and female-specialist colonies of the fire ant Solenopsis invicta. These exchanges quickly reversed the sex-ratio biases of adopting colonies. The sex ratio of queen-laid eggs differed strongly between male- and female-specialist colonies. These findings suggest that queens can force workers to raise male sexuals by limiting the number of female brood and help to explain why sex investment ratios lie between the queen and worker equilibria in this and many other ant species.

Wolbachia infections in native and introduced populations of fire ants (Solenopsis spp.).

Wolbachia are cytoplasmically inherited bacteria that induce a variety of effects with fitness consequences on host arthropods, including cytoplasmic incompatibility, parthenogenesis, male-killing and feminization. We report here the presence of Wolbachia in native South American populations of the fire ant Solenopsis invicta, but the apparent absence of the bacteria in introduced populations of this pest species in the USA. The Wolbachia strains in native S. invicta are of two divergent types (A and B), and the frequency of infection varies dramatically between geographical regions and social forms of this host. Survey data reveal that Wolbachia also are found in other native fire ant species within the Solenopsis saevissima species complex from South America, including S. richteri. This latter species also has been introduced in the USA, where it lacks Wolbachia. Sequence data reveal complete phylogenetic concordance between mtDNA haplotype in S. invicta and Wolbachia infection type (A or B). In addition, the mtDNA and associated group A Wolbachia strain in S. invicta are more closely related to the mtDNA and Wolbachia strain found in S. richteri than they are to the mtDNA and associated group B Wolbachia in S. invicta. These data are consistent with historical introgression of S. richteri cytoplasmic elements into S. invicta populations, resulting in enhanced infection and mtDNA polymorphisms in S. invicta. Wolbachia may have significant fitness effects on these hosts (either directly or by cytoplasmic incompatibility) and therefore these microbes potentially could be used in biological control programmes to suppress introduced fire ant populations.

Hierarchical analysis of genetic structure in native fire ant populations: results from three classes of molecular markers.

We describe genetic structure at various scales in native populations of the fire ant Solenopsis invicta using two classes of nuclear markers, allozymes and microsatellites, and markers of the mitochondrial genome. Strong structure was found at the nest level in both the monogyne (single queen) and polygyne (multiple queen) social forms using allozymes. Weak but significant microgeographic structure was detected above the nest level in polygyne populations but not in monogyne populations using both classes of nuclear markers. Pronounced mitochondrial DNA (mtDNA) differentiation was evident also at this level in the polygyne form only. These microgeographic patterns are expected because polygyny in ants is associated with restricted local gene flow due mainly to limited vagility of queens. Weak but significant nuclear differentiation was detected between sympatric social forms, and strong mtDNA differentiation also was found at this level. Thus, queens of each form seem unable to establish themselves in nests of the alternate type, and some degree of assortative mating by form may exist as well. Strong differentiation was found between the two study regions using all three sets of markers. Phylogeographic analyses of the mtDNA suggest that recent limitations on gene flow rather than longstanding barriers to dispersal are responsible for this large-scale structure.

Juvenile hormone in adult eusocial Hymenoptera: gonadotropin and behavioral pacemaker.

Studies on the role of juvenile hormone (JH) in adult social Hymenoptera have focused on the regulation of two fundamental aspects of colony organization: reproductive division of labor between queens and workers and age-related division of labor among workers. JH acts as a gonadotropin in the primitively eusocial wasp and bumble bee species studied, and may also play this role in the advanced eusocial fire ants. However, there is no evidence that JH acts as a traditional gonadotropin in the advanced eusocial honey bee or in the few other ant species that have recently begun to be studied. The role of JH in age-related division of labor has been most thoroughly examined in honey bees. Results of these studies demonstrate that JH acts as a "behavioral pacemaker," influencing how fast a worker grows up and makes the transition from nest activities to foraging. Hypotheses concerning the evolutionary relationship between the two functions of JH in adult eusocial Hymenoptera are discussed.

Social evolution in a new environment: the case of introduced fire ants.

The inadvertent introduction of the fire ant Solenopsis invicta to the United States from South America provides the opportunity to study recent social evolution by comparing social organization in native and introduced populations. We report that several important elements of social organization in multiple-queen nests differ consistently and dramatically between ants in Argentina and the United States. Colonies in Argentina contain relatively few queens and they are close relatives, whereas colonies in the United States contain high numbers of unrelated queens. A corollary of these differences is that workers in the native populations are significantly related to the new queens that they rear in contrast to the zero relatedness between workers and new queens in the introduced populations. The observed differences in queen number and relatedness signal a shift in the breeding biology of the introduced ants that is predicted on the basis of the high population densities in the new range. An additional difference in social organization that we observed, greater proportions of permanently unmated queens in introduced than in native populations, is predicted from the loss of alleles at the sex-determining locus and consequent skewing of operational sex ratios in the colonizing ants. Thus, significant recent social evolution in fire ants is consistent with theoretical expectations based on the altered ecology and population genetics of the introduced populations.

Effect of a founder event on variation in the genetic sex-determining system of the fire ant Solenopsis invicta.

Effects of a recent founder event on genetic diversity in wild populations of the fire ant Solenopsis invicta were studied, with particular attention given to the genetic sex-determining system. Diploid males are far more common relative to haploid males in introduced populations than in native populations of fire ants, and queens that produce diploid males account for a significantly larger proportion of the mated queens in introduced than in native populations. Differences between native and introduced populations in attributes of the mating systems (i.e., queen mating frequency or level of inbreeding) can be excluded as factors contributing to these different levels of diploid male production. Thus, we conclude that diploid males have increased in frequency in introduced populations because of a loss of allelic diversity at the sex-determining locus (loci). This loss of sex alleles has generated a substantial increase in the estimated segregational genetic load associated with production of sterile diploid males in introduced populations over the load in native populations. The loss of allelic diversity in the sex-determining system in introduced S. invicta is paralleled by a loss of electrophoretically detectable rare alleles at protein-encoding loci. Such concordance between these different types of markers is predicted because each of the many sex alleles present in the native populations is expected to be rare. Estimates of expected heterozygosity (Hexp) based on 76 electrophoretic loci do not differ significantly between the native and introduced fire ant populations, illustrating the lack of sensitivity of this measure for detecting many types of bottlenecks.