Wolbachia causes cytoplasmic incompatibility but not male‐killing in a grain pest beetle

The endosymbiotic Wolbachia is one of the most common intracellular bacteria known in arthropods and nematodes. Its ability for reproductive manipulation can cause unequal inheritance to male and female offspring, allowing the manipulator to spread, but potentially also impact the evolutionary dynamics of infected hosts. Estimated to be present in up to 66% of insect species, little is known about the phenotypic impact of Wolbachia within the order Coleoptera. Here, we describe the reproductive manipulation by the Wolbachia strain wSur harboured by the sawtoothed grain beetle Oryzaephilus surinamensis (Coleoptera, Silvanidae), through a combination of genomics approaches and bioassays. The Wolbachia strain wSur belongs to supergroup B that contains well‐described reproductive manipulators of insects and encodes a pair of cytoplasmic incompatibility factor (cif) genes, as well as multiple homologues of the WO‐mediated killing (wmk) gene. A phylogenetic comparison with wmk homologues of wMel of Drosophila melanogaster identified 18 wmk copies in wSur, including one that is closely related to the wMel male‐killing homologue. However, further analysis of this particular wmk gene revealed an eight‐nucleotide deletion leading to a stop‐codon and subsequent reading frame shift midsequence, probably rendering it nonfunctional. Concordantly, utilizing a Wolbachia‐deprived O. surinamensis population and controlled mating pairs of wSur‐infected and noninfected partners, we found no experimental evidence for male‐killing. However, a significant ~50% reduction of hatching rates in hybrid crosses of uninfected females with infected males indicates that wSur is causing cytoplasmic incompatibility. Thus, Wolbachia also represents an important determinant of host fitness in Coleoptera.

to be present in up to 66% of insect species, little is known about the phenotypic impact of Wolbachia within the order Coleoptera. Here, we describe the reproductive manipulation by the Wolbachia strain wSur harboured by the sawtoothed grain beetle Oryzaephilus surinamensis (Coleoptera, Silvanidae), through a combination of genomics approaches and bioassays. The Wolbachia strain wSur belongs to supergroup B that contains well-described reproductive manipulators of insects and encodes a pair of cytoplasmic incompatibility factor (cif) genes, as well as multiple homologues of the WO-mediated killing (wmk) gene. A phylogenetic comparison with wmk homologues of wMel of Drosophila melanogaster identified 18 wmk copies in wSur, including one that is closely related to the wMel male-killing homologue. However, further analysis of this particular wmk gene revealed an eight-nucleotide deletion leading to a stop-codon and subsequent reading frame shift midsequence, probably rendering it nonfunctional. Concordantly, utilizing a Wolbachia-deprived O. surinamensis population and controlled mating pairs of wSur-infected and noninfected partners, we found no experimental evidence for male-killing. However, a significant ~50% reduction of hatching rates in hybrid crosses of uninfected females with infected males indicates that wSur is causing cytoplasmic incompatibility. Thus, Wolbachia also represents an important determinant of host fitness in Coleoptera.

| INTRODUC TI ON
Symbiotic bacteria influence the ecology and evolution of animals in various ways (Douglas, 2015;McFall-Ngai et al., 2013). Insects harbour an especially high abundance and diversity of microbial associations that span the entire range from parasitism to mutualism (Drew et al., 2021). While some symbionts exhibit a very strict phenotype, others incur context-dependent impacts along the parasitemutualist continuum including host fitness benefits or costs (Feldhaar, 2011;Oliver & Martinez, 2014;Zytynska et al., 2021).
However, a large proportion of insects are also infected by multiple symbionts that each on their own exhibit different, stable phenotypes, such as nutritional supplementation and reproductive manipulation, but could interfere with each other (Duron et al., 2008). In consequence, host ecology and evolution can be driven by multiple symbionts with possibly different selective interests.
Wolbachia bacteria (α-Proteobacteria) are some of the most common intracellular bacteria known in arthropods and nematodes (Werren et al., 2008). They are predominantly parasitic and transmitted maternally between host generations, but horizontal transmission occurs occasionally. Wolbachia employ several distinct strategies to maximize their transmission by influencing the germ line of their host. Thereby, they can rapidly sweep through uninfected populations and then maintain a high prevalence within a population. These mechanisms include cytoplasmic incompatibility (CI), parthenogenesis, male-killing or feminization (Werren et al., 2008).
While CI leads directly to a higher proportion of infected individuals, the other mechanisms lead to a higher proportion of female individuals in the population. This in turn increases the fitness of Wolbachia, which is predominantly transmitted maternally (Heath et al., 1999).
However, Wolbachia infection does not necessarily result in reproductive manipulation with negative fitness consequences for the host (Zug & Hammerstein, 2015). Furthermore, Wolbachia can even evolve into a mutualist and enhance its host's fitness by supplementing dietary-limited nutrients, such as B-vitamins like riboflavin (Hosokawa et al., 2010;Ju et al., 2019;Moriyama et al., 2015).
CI and male-killing are the predominant strategies of reproductive manipulation in insects (Fialho & Stevens, 2000;Perlmutter et al., 2020). CI generally refers to factors localized in the cytoplasm of sperm and eggs that render them incompatible with each other, resulting in inviable embryos (Beckmann et al., 2019;. Wolbachia causes CI by expressing a "killing" factor in the male sperm. In eggs of uninfected females, this modification leads to nonviable embryos, whereas in infected females a "rescue" factor reverses this modification so that the zygote can develop normally (Shropshire, 2020). While unidirectional CI occurs when infected males mate with uninfected females resulting in fertilized but unviable eggs, bidirectional CI occurs when two individuals are infected by different, yet incompatible Wolbachia strains (Werren et al., 2008). Recently, two cytoplasmic incompatibility factor genes (cifA and cifB) have been identified as key factors in CI-inducing Wolbachia strains (LePage et al., 2017).
The pair of CI-inducing genes were not found in the chromosomal Wolbachia genes, but in the integrated eukaryotic association module of phageWO (LePage et al., 2017). A two-by-one genetic model has been suggested, specifying that while both cifA and cifB induce CI, only cifA is able to rescue the CI phenotype when transgenically expressed in the host's ovaries (Shropshire et al., 2018;. The other widespread phenotype of Wolbachia inducing reproductive manipulation is male-killing. During embryogenesis, the development of the male embryo is disturbed by Wolbachia, leading to embryonic lethality (Werren et al., 2008). In consequence, the fitness of infected sister embryos is enhanced by higher allocation of resources during ovogenesis and reduced intraspecific competition during juvenile development and adult life (Hurst & Jiggins, 2000;Jaenike et al., 2003). The gene WO-mediated killing (wmk) of the Wolbachia strain wMel of the fruit fly Drosophila melanogaster has been identified to recapitulate this male-killing phenotype when transgenically expressed in D. melanogaster flies (Perlmutter et al., 2019). So far, wmk homologues have been found in all Wolbachia strains associated with male-killing, surprisingly also localized within the eukaryotic association module of phageWO, only a few genes upstream from the CI-inducing genes cifA and cifB (Perlmutter et al., 2019). There are at least five homologues of the wmk gene encoded in the genome of wMel and the function of many of these remain enigmatic as only the transgenic expression of the original wmk gene, but not other homologues, caused male-killing (Perlmutter et al., 2020). Wolbachia strains causing CI and male-killing phenotypes have been well studied within the insect orders Diptera and Hymenoptera, such as the fruit fly D. melanogaster (Perlmutter et al., 2020), the southern house mosquito Culex quinquefasciatus (Duron et al., 2005) and the parasitoid wasp Nasonia vitripennis (Bordenstein & Werren, 1998, 2007. Although beetles infected with Wolbachia have repeatedly been reported in recent years, little is known about the functional consequences of Wolbachia infections within the order Coleoptera (Aikawa et al., 2022;Fialho & Stevens, 2000;Heddi et al., 1999;Kajtoch & Kotásková, 2018;Li et al., 2015;Li et al., 2016).
The endosymbiont S. silvanidophilus provides aromatic amino acid precursors for cuticle synthesis of the host via the shikimate pathway (Kiefer et al., 2021). In addition, O. surinamensis is commonly infected with Wolbachia (Li et al., 2015;Sharaf et al., 2010). Sharaf et al. (2010) identified a higher Wolbachia infection rate in feral populations of O.
surinamensis compared to adapted silo populations, but also a strong female bias among adults emerging under laboratory conditions, suggesting active reproductive manipulation by these Wolbachia strains.
Elucidating Wolbachia's capabilities of reproductive manipulation in O.
surinamensis is therefore relevant in understanding the biology of this agricultural pest as well as a symbiotic model insect.
In this work, we localized Wolbachia in the O. surinamensis JKI strain and quantified its growth dynamics across developmental stages. A phylogenetic analysis and functional prediction of the associated Wolbachia wSur genome revealed it to be a member of supergroup B, presumably capable of CI as it encodes homologues of the cytoplasmic incompatibility factor genes cifA and cifB. However, the strain is incapable of inducing male-killing, possibly due to an eight-nucleotide deletion in the identified male-killing gene wmk creating a stop codon as well as subsequent reading frame-shift. Finally, we experimentally tested the predicted phenotype of reproductive manipulation-unidirectional CI and no male-killing-using mating assays of beetles with manipulated infection status, where we were able to verify the phenotype of reproductive manipulation via unidirectional CI.

| Insect cultures
The initial Oryzaephilus surinamensis culture (strain JKI) was obtained from the Julius-Kühn-Institute/Federal Research Centre for Cultivated Plants in 2014 and kept in culture since then. Continuous symbiotic and aposymbiotic (by aposymbiotic we refer in this paper to beetles without both S. silvanidophilus and wSur symbionts). O.
surinamensis cultures (see below) were maintained in 1.8-L plastic containers, filled with 50 g oat flakes, at 28°C, 60% relative humidity and a day and night cycle of 16/8 h.

| Elimination of O. surinamensis symbionts
An O. surinamensis sub-population was treated for 12 weeks with tetracycline (150 mg/5 g oat flakes, see for details see  to eliminate both of their symbionts (S. silvanidophilus and wSur) and then kept for several generations on a normal diet to exclude direct effects of tetracycline on the host physiology. A control group was established in parallel with all steps except the addition of tetracycline to account for any unforeseen effects of the handling, population bottlenecks, etc. The apo−/symbiotic status regarding both symbionts of these beetle sub-populations was confirmed before each following experiment. Therefore, female adult beetles were individually separated in single jars with oat flakes to lay eggs.
After 4 weeks, the adult generation was removed before their offspring finished metamorphosis, DNA of these parent females was extracted and the symbiont titre was analysed by quantitative PCR (polymerase chain reaction; see below).
CFB563_rev (5′-GCACCCTTTAAACCCAAT-3′) Kiefer et al., 2021). qPCR was carried out on a Rotor-Gene Q thermal cycler (Qiagen). The initial temperature was 95°C for 12 min, followed by 60 cycles of 95°C for 40 s followed by 20 s at 60°C. A melting curve analysis was used to assess the specificity of the qPCR reaction by a gradual increase of temperature from 60 to 95°C, with 0.25°C/s. The qPCR results were analysed using the Rotor Gene Q Software (Qiagen).
Standard curves with defined copy numbers of the 16 S rRNA gene were created by amplifying the fragment first via PCR using the previously mentioned primers, followed by purification via an innuPREP PCRpure (Analytik Jena) and determination of the DNA concentration via a NanoDrop1000 (Peqlab). After determination of the DNA concentration, a standard containing 10 10 copies/μl was generated and 1:10 serial dilutions down to 10 1 copies/μl were prepared. One microlitre of each standard was included in a qPCR to standardize all measurements.

| Symbiont genome sequencing, assembly and annotation
We

| Identifying genes important for reproductive manipulation
The obtained genome was manually searched for wmk, cifA and were aligned using muscle (Edgar, 2004) in geneious prime 2019 (version 2019.1.3; https://www.genei ous.com). In addition, we reanalysed a set of sequencing libraries from O. surinamensis sampled in a grain storage facility and two field sites in Israel (SRX2583549-SRX2583574) . We assembled reads following the workflow of our own data set and extracted wmk homologues from Wolbachia contigs by searching for genes annotated as "Transcriptional regulator" as well as mapping wmk homologues from the JKI wSur strain against the assemblies and vice versa. All wmk homologues from the first analysis were combined with wmk1 and wmk12-like homologues from all these strains and aligned as mentioned above.
Phylogenetic reconstructions of the nucleotide alignment were performed with the MrBayes-plugin (Huelsenbeck & Ronquist, 2001) of geneious prime using the HKY85 substitution model and invgamma rate variation as recommended by jmodeltest 2.1.10 version 20,160,303 (Sullivan et al., 2012). The analysis ran for 1,100,000 generations, with a burn-in of 100,000 generations and trees sam-

| Bioassays for reproductive manipulation
By mating experiments with differentially infected individuals, we The isolated individuals were observed until hatching, and the sex of the individual insect was determined by the presence (males) or absence (females) of spikes on the third femur (Halstead, 1963). Males and females were combined into mating pairs at an age of 7-10 days.
In total, 30 mating pairs were prepared, 10 for each group: The first group consisted of mating pairs where both partners, female and male, were aposymbiotic, whereas the second group was made up of crossings with two symbiotic partners. The third group contained symbiotic males of O. surinamensis paired with aposymbiotic females. The mating pairs were given one microspatula scoop of ground oat previously filtered through a 0.6-mm sieve. Furthermore, to prevent the specimens escaping the setup, the edge of each individual well was coated with a polytetrafluoroethylene 60 wt% dispersion in H 2 O (PTFE-dispersion; Sigma-Aldrich). For the first 2 weeks of the experiment all pairs were left undisturbed. In the following 6-week period, the number of laid eggs and hatched larvae were counted twice weekly, and the adults were placed one well further down in the 24-well plate. In addition, we quantified the sex ratio of 100 randomly picked individuals in both symbiotic and aposymbiotic stock cultures to test for a sex bias induced by male-killing.

| Statistical procedure for qPCR results and differences in hatching rate and sex ratio
The influence of glyphosate and tetracycline on the symbiont titre of the adult beetles ( Figure S1) was analysed using Dunn's test from the package "FSA" in rstudio (R version 4.1.1) with two-sided post hoc tests corrected for multiple testing using the Benjamini-Hochberg (BH) method (Benjamini & Hochberg, 1995;Dunn, 1964). A compact letter display (CLD; Piepho & Piepho, 2009) was generated with the package "rcompanion" (Mangiafico, 2017). Comparison between hatching rates was performed with Wilcoxon rank sum tests including correction for false discovery rates (FDRs) by repeated testing following the BH procedure (Benjamini & Hochberg, 1995), implemented in the R package "stats." Plots were visualized using "gg-plot2" (Wickham, 2016). Sex ratio in beetle cultures was analysed using a manually calculated χ 2 test of homogeneity. imum as early as in the pupa during early metamorphosis (early pupa: 5.9 × 10 6 median copies; late pupae: 3.9 × 10 6 median copies; Figure 2, left), while we observed in the same sample set a peak of S. silvanidophilus only within the first week after metamorphosis (male 6.7 × 10 7 median copies and female 6.7 × 10 7 median copies;  Figure S1). While strict tetracycline treatment eliminated both S. silvanidophilus Kiefer et al., 2021) and wSur (Kruskal-Wallis χ 2 = 52.605, df = 7, p = .000000004437, Dunn's test: p < .05; Figure S1), resulting in dual aposymbiotic (hereafter aposymbiotic) beetles, the herbicide glyphosate had a differential effect: S. silvanidophilus was drastically reduced, yet still present in low amounts while wSur was not negatively affected (Kruskal-Wallis χ 2 = 52.605, df = 7, p = .000000004437, but Dunn's test: p > .05; see Table S1 for pairwise comparisons; Figure S1).

| Genomics and phylogeny of the Wolbachia strain
We previously sequenced the metagenome of O. surinamensis combining short-and long-read technologies (Illumina and ONT) into a hybrid assembly. Besides the Bacteroidota endosymbiont S. silvanidophilus (Kiefer et al., 2021) we also extracted the full genome of a Wolbachia strain in a single, circular contig in the assembly (Figure 3).
The circular genome is 1,728,764 bp in length with an average GC content of 34.1% and a coverage of 186× with short-read sequences F I G U R E 1 Fluorescence in situ hybridization micrographs of Wolbachia (green) and Shikimatogenerans (magenta) in sagittal sections of (a) a 5-dayold Oryzaephilus surinamensis pupa stained with a Bacteroidota-specific probe highlighting Shikimatogenerans silvanidophilus (CFB563mod-Cy3, magenta), and in the gonads of (b) an adult female and (c) an adult male stained with a eubacteria-specific probe highlighting S. silvanidophilus (EUB338-Cy3, magenta), Wolbachia-specific probes (Wol-W3-Cy5 and Wolb-2-Cy5, green) and DAPI targeting DNA in general (white). b = bacteriomes, c = cuticle, o = ovariole, t = testes, sv = seminal vesicle. Image (a) was originally published without the Wolbachia channel in Kiefer et al. (2021). wSur Shikimatogenerans silvanidophilus  Table 1).
Besides general genetic information processing including DNA replication and repair, transcription, and translation, the genome also contained a full glycolysis pathway to process glucose-6phosphate to erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP). Further, it contained a full riboflavin pathway and the pathways to synthesize the amino acids lysine, glutamine, threonine, glycine, and serine but no single gene of the shikimate pathway to synthesize aromatic amino acids, explaining its insensitivity to glyphosate (Fischer et al., 1986;Gresshoff, 1979;Steinrücken & Amrhein, 1980).

| Analysis of male-killing gene candidates
The genome of wSur contained seven regions with phage WOassociated genes (WOSurA-WOSurG) in total, each with two to three homologues of the wmk gene ( Figure 3). Overall, the genome coded for 18 wmk homologues which were numbered from wmk1 to wmk18. As these copies may share the ability to induce malekilling, we compared these wmk homologues of wSur with the functionally described wmk homologues in the wMel strain as well as other known male-killing strains. Phylogenetic analysis identified homologues wmk1 and wmk12 in the phage region WOSurB as the most likely candidates to confer male-killing due to their high sequence similarity with the functional homologue wmk in wMel (for wmk12), as well as wInn and wBor (for wmk1; Figure 5 Figure S2). In addition, we only found wmk1-like homologues in individuals from the grain storage population, but in no individual from the feral populations.
We tested for symbiont-mediated male-killing phenotype in the χ 2 test of homogeneity: χ 2 = 0.080, p = .888). In addition, the malekilling phenotype should also result in a reduced hatching rate of around 50% in mating pairs with symbiotic females and males in comparison with mating pairs with aposymbiotic individuals. However, we did not observe such differences (BH-corrected Wilcoxon rank sum test, p = .84; Figure 6, right).

| Cytoplasmic incompatibility (CI)
Single homologues of both previously identified CI factor genes We tested the ability of Wolbachia infection to cause cytoplasmatic incompatibility by mating experiments with differential wSur infection. First, the impact of Wolbachia infection on the number of laid eggs was determined. As expected, infection with wSur had no effect on the number of laid eggs (Kruskal Wallis test: χ 2 = 0.29, df = 2, p = .86; Figure 6, left). Following further development, we observed overall differences between the three groups' hatching rates (Kruskal Wallis test: χ 2 = 10.85, df = 2, p = .004397; Figure 6, right). While the hatching rate between the control groups did not differ (aposymbiotic females and males, as well as symbiotic females and males: BH-corrected Wilcoxon rank sum test: p = .84; Figure 6, right), the hatching rate in the CI cross with aposymbiotic females and symbiotic males right was reduced by 43%-47% in comparison to both control groups (BH-corrected Wilcoxon rank sum test, p = .04 and .0018; Figure 6).  (Arkin et al., 2018), based on the fasttree2 algorithm (Price et al., 2010). Node numbers represent local support values. RefSeq assembly accession numbers are given in square brackets. The supergroups are colour-coded and indicated on the right (Baldo et al., 2006;Bandi et al., 1998;Casiraghi et al., 2005;Werren et al., 1995). The Wolbachia endosymbiont of Oryzaephilus surinamensis (wSur, highlighted in red font) belongs to supergroup B. Wolbachia strain genomes highlighted in bold font were utilized for subsequent phylogenetic analyses of wmk ( Figure 5) and cif genes (Figure 7). Drosophila melanogaster which were proposed as candidate genes responsible for the induction of Wolbachia's male-killing phenotype (Perlmutter et al., 2019(Perlmutter et al., , 2020.

Multiple homologues of wmk have been described in other
Wolbachia strains, although all except one did not induce malekilling when transgenically expressed in D. melanogaster (Perlmutter et al., 2020). Currently, the function of the additional wmk homologues in wSur, as well as wMel, remains unknown. wSur and other strains might be multipotent and capable of inducing male-killing under specific conditions, or when infecting other hosts, such as the Wolbachia strain wRec inducing CI in its main host Drosophila recens but causing male-killing when transferred to the closely related species Drosophila subiquinaria (Jaenike, 2007). In addition, the Wolbachia strains might manipulate the host in different ways beyond reproductive manipulation, for example by affecting pheromone biosynthesis, perception or behaviour (Bi & Wang, 2020;Farahani et al., 2021;Schneider et al., 2019).
The wmk12/1 duplication in wSur at least suggests that it is benefi- as type I to type V (Bing et al., 2020;LePage et al., 2017;Lindsey et al., 2018). The CI phenotype was demonstrated in cif genes of type I, II and IV (LePage et al., 2017). Our analysis classified the cifA of wSur as a type II homologue, while cifB clustered between type I and II homologues. Our experimental data indicate wSur to be a reproductive manipulator by causing unidirectional CI to its host. Crossing Wolbachia-infected males with uninfected females resulted in a hatching rate that was reduced by 45% compared to crossings between infected males and females or uninfected males and females, respectively. Findings in Drosophila simulans showed a strong induction of CI leading to a hatching rate reduction of up to 95% (Sinkins et al., 1995), while data from D. melanogaster showed weak induction of CI resulting in a hatching rate reduced by 15%-30% (Hoffmann et al., 1994), depending on environmental conditions (Hague et al., 2020) Figure S1). Thus, S. silvanidophilus is responsible for supplementation of cuticle synthesis as well as ecological consequences in terms of elevated resistance to abiotic desiccation stress, pathogen and predation pressure (Kanyile et al., 2022), but also costs of symbiont infection on reproduction (Engl et al., 2020).
Certain Wolbachia strains were previously reported to supplement the hosts' diet with limited nutrients (especially B-vitamins; Hosokawa et al., 2010) or provide pathogen defence (Moreira et al., 2009). The Wolbachia strain wSur of O. surinamensis also encodes pathways to synthesize the amino acids lysine, glutamine, threonine, glycine and serine as well as the vitamin riboflavin.
It remains unclear whether Wolbachia might synthesize lysine only for its own benefit, or also contribute it to its host's metabolism.
Similarly, it is unclear whether Wolbachia infection inflicts additional costs beyond unidirectional CI which is only relevant in populations with incomplete Wolbachia infection (Hoffmann et al., 1996;Perrot-Minnot et al., 2002;Vala et al., 2000).
However, currently both symbioses are usually studied by experimental approaches in isolation, or from a descriptive perspective on the prevalence and genomic potential. Thereby, we miss out on potential higher levels of ecological interactions of both types of symbioses, mediated either via the host's physiology, or even directly between different symbionts. Future work should thus try to integrate multipartite, symbiotic relationships. Available tools include selective removal or inhibition of individual symbionts, such as by targeting specific, obligate biosynthetic pathways of symbionts. The glyphosate utilized here, inhibiting the Shikimate pathway responsible for the synthesis of aromatic amino acids (Steinrücken & Amrhein, 1980), but, for example, also inhibitors of the diaminopimelate pathway responsible for synthesizing lysine are prominent agents suggested for the manipulation of specific biosynthetic capabilities or organisms encoding them (Hutton et al., 2003). F I G U R E 7 Phylogeny and domain structure of cif genes. Top: Bayesian phylogenies based on a nucleotide alignment of cifA (top) and cifB (middle) genes. Consensus support values are shown at the branches. Coloured shapes around branches designate monophyletic "types." bottom: Domain structure for the cif genes of wSur. The two loci (PD-[D/E]XK nuclease/DpnII-MboI protein domain) of cifB are shown and indicated with orange bars.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Genetic data: Raw sequence reads are deposited in the SRA (SRR12881563-SRR12881566; SRR12881567-SRR12881568; BioProject PRJNA670819). The annotated wSur genome is available on GenBank (CP092526). Bioassay data are available on the data repository of the Max-Planck-Society Edmond .

B EN EFIT-S H A R I N G S TATEM ENT
All specimens utilized in this work were obtained from a longstanding laboratory culture (before 2014). Thus, the Nagoya Protocol is not applicable and no benefits are reported.