Identification and pathogenicity of Pratylenchus scribneri on tomato in Sichuan Province of People's Republic of China

Root-lesion nematodes (Pratylenchus spp.) are a group of economically important pathogens that have caused serious economic losses in many crops. In 2019, root-lesion nematodes were recovered from tomato (Solanum lycopersicum) root samples collected from Sichuan Province, People's Republic of China (PRC). Extracted nematodes were disinfected, and one individual female was cultured on a carrot disc for propagation at 25 °C by parthenogenesis and designated the SC isolate. Afterwards, the isolate was identified on the basis of morphometric and molecular markers. Both morphometric characters and molecular analysis of the internal transcribed spacer region gene (ITS) of ribosomal DNA, the D2-D3 expansion region of the 28S rDNA gene and the mitochondrial cytochrome oxidase I (mtDNA-COI) gene revealed that the species of root-lesion nematode was Pratylenchus scribneri. The Bayesian tree inferred from the ITS rDNA, 28S rDNA and mtDNA-COI gene sequences also showed that this isolate formed a highly supported clade with other P. scribneri isolates. The pathogenicity of the root-lesion nematode SC isolate on tomato was assessed, showing that tomato was a suitable host for P. scribneri. To the best of our knowledge, this is the first report of P. scribneri on tomato in Sichuan Province, PRC. These are also the first molecular data obtained from P. scribneri on tomato in the PRC, and the pathogenicity of P. scribneri to tomato was studied for the first time. This study provides scientific data for the detection, identification and control of tomato root-lesion nematode disease.

The genus Diaphanosoma (Diplostraca: Sididae) in Greece: morphological and molecular assessment

For the genus Diaphanosoma Fischer (Cladocera) the species’ name “D. brachyurum” has been widely used for many decades to identify other species belonging to this genus. To clarify the diversity of the genus in Greek lakes in the present study, we morphologically and genetically identified the Diaphanosoma species occurring in eight lakes. Three hundred twenty-nine Diaphanosoma individuals were morphologically examined, while for the genetic analyses the mtDNA COI gene was sequenced in 48 individuals. Combining the morphological and genetic results, we verified the occurrence of D. mongolianum, D. orghidani and D. macedonicum in our study area. We could not confirm prior records of D. brachyurum and D. lacustris while we provide the molecular identity of D. macedonicum. Furthermore, we highlight the need to check whether the European D. mongolianum populations are characterised of mitochondrial discordance and hybridization as the individuals from the Asian type locality of the species. Our results support the importance of combining both approaches to correctly identify taxonomic species, despite the extra effort and cost during the sample analysis.

Integrative Taxonomy of Two Peruvian Strains of Brachionus plicatilis Complex with Potential in Aquaculture

Two Peruvian strains of the genus Brachionus were isolated from impacted coastal wetlands. With an integrative taxonomic view, we described their taxonomic status, morphological characters, productive parameters, and phylogenetic position. In the case of both strains, the relationship between biometrics and productive parameters obtained with Principal Components Analysis indicated that the lorica length was associated with longevity, progeny, egg production, and reproductive age, while the lorica width and aperture were associated with the maximum number of eggs carried. Maximum Likelihood and Bayesian Inference analysis carried out with mtDNA COI gene and rDNA ITS1 region showed that both strains were clustered in two clades with distinct phylogenetic positioning from what is currently known for Brachionus plicatilis s.l. One of the strains, Z010-VL, is proposed to be a subspecies of L4 (B. paranguensis), and the other strain, Z018-SD, is proposed as a sub species of SM2 (B. koreanus). In addition, 33 and 31 aquaculture production lineages are proposed, delimited by COI and concatenated COI+ITS1 sequences, respectively. Finally, this study provides new tools that enhance the traceability of the origin of each sub-species throughout the world.

Unusual Morphotypes of the Giant Barrel Sponge off the Coast of Barbados

Giant barrel sponges (GBSs) belong to a cryptic species complex (Xestospongia spp.) and are found on tropical reefs worldwide. Over their range, including most of the Caribbean, GBSs have a cylindrical shape, with variation in height, diameter and surface complexity. However, off the southwest coast of Barbados, GBSs mostly exhibit a clam shape or a tub shape, interspersed with a few that have the normal barrel morphotype, suggesting that this variation is not due to environmental factors. Haplotype identification (mtDNA-COI) of six clam and six normal sponges indicated no clear genetic differentiation based on morphotype; hence, this morphological variation remains unexplained.

High genetic diversity of bivalve transmissible neoplasia in the blue mussel Mytilus trossulus Gould from the subarctic Sea of Okhotsk

There are increasing findings of the bivalve transmissible neoplasia derived from the Pacific mussel Mytilus trossulus (MtrBTN) in populations of different Mytilus species worldwide. The Subarctic is an area where this disease has not yet been sought despite the fact that Mytilus spp. are widespread there, and M. trossulus itself is a boreal species. We used cytological and histological techniques to diagnose disseminated neoplasia in a sample of M. trossulus from Magadan in the subarctic Sea of Okhotsk. Neoplasia was identified in 11 of 214 mussels studied. Using mtDNA COI sequencing, we revealed genotypes identical or nearly identical to known MtrBTN ones in the hemolymph of most of the diseased mussels. Both MtrBTN evolutionary lineages have been identified, the widespread MtrBTN2, and MtrBTN1, so far only known from M. trossulus in British Columbia on the other side of the Pacific from Magadan. In addition, MtrBTN2 was represented by two common diverged mtDNA haplolineages. These conclusions have been confirmed for selected cancerous mussels by molecular cloning of COI and additional nuclear and mtDNA genes. On the background of high genetic diversity, different cancers were similar in terms of ploidy (range 4.0 - 5.8n) and nuclear to cell ratio. Our study provides the first description of neoplasia and MtrBTN in mussels from the Sea of Okhotsk and from the Subarctic, of both MtrBTN1 and MtrBTN2 in the same mussel population, and the first direct comparison between these transmissible cancers.

Distribution of Two Strains of Leptoglossus zonatus (Dallas) (Hemiptera: Coreidae) in the Western Hemisphere: Is L. zonatus a Potential Invasive Species in California?

The leaffooted plant bug, Leptoglossus zonatus (Dallas) (Hemiptera: Coreidae) is polyphagous and widely distributed in the Western Hemisphere. Although it has been recorded in California since around 1900, it has become a more common pest in almonds in the last decade. Other studies have shown that an established insect can become a pest when a new genotype is introduced. This study investigated the distribution of two lineages (strains) of L. zonatus in the Western Hemisphere. Specimens from the Leptoglossus collection in the national insect collection in Mexico were used to extract DNA and sequence the mitochondrial DNA cytochrome oxidase I (mtDNA COI) gene, for use in population genetic and phylogenetic analyses. New sequences from Mexico, Central and South America were combined with those available in GenBank, from California and Brazil. Two lineages (strains) of L. zonatus were uncovered. One lineage occurs in California, Mexico and Ecuador. The second lineage is more widespread and found in California, Mexico, Guatemala, Nicaragua, Bolivia and Brazil. The haplotype number and diversity, and nucleotide diversity, were found for samples from California, Mexico, and Brazil, for the two lineages, and for all 118 sequences combined. All sequences combined produced five haplotypes, and a haplotype diversity of 0.54. California and Brazil had 3 haplotypes each, with one haplotype shared (5 total). Haplotype diversity in California and in Brazil were 0.526 and 0.505, respectively. A haplotype network found that one haplotype was most abundant and widespread. The small number of haplotypes, a range expansion, and economic pest status of L. zonatus in California, all contribute to this insect being a potentially invasive insect pest.

The Adult, Larva, and Pupa of a New Pseudopyrochroa (Coleoptera: Pyrochroidae: Pyrochroinae) from China, with Molecular Phylogenetic Inferences

A new species of Pseudopyrochroa Pic, 1906, P. reni Pan & Young, n. sp., is described from the western region of Mt. Qinling, China. Larvae, pupae, and adults were associated using molecular phylogenetic analyses based on mtDNA COI barcode sequences. All three stages are described and illustrated. Additionally, preliminary phylogenetic relationships among five genera and 14 species of Pyrochroidae, including Pseudopyrochroa, are hypothesized based on COI sequence data. The fauna of Pyrochroidae from the Mt. Qinling biodiversity conservation area is discussed.

First Report of Uncinaria hamiltoni in Orphan Eastern Mediterranean Monk Seal Pups in Greece and Its Clinical Significance

The Mediterranean monk seal (Monachus monachus) is classified by the IUCN as “endangered,” with a global population estimated to number fewer than 800 individuals. Our understanding of the biology and health status of the species is still limited, rendering every medical case a challenge for conservationists and veterinary clinicians. Although studying and managing disease in wild marine hosts is complex and challenging, studying and mitigating the effects of any disease to the Mediterranean monk seal is of utmost importance for conservation. The aim of this study was to document for the first time the presence of the hookworm Uncinaria hamiltoni in rehabilitated Mediterranean monk seal pups in Greece. A detailed examination protocol was followed for all pups that live-stranded over 30 years in 22 different locations, including physical, parasitological, and other examinations. Hookworms (adults and/or eggs) were detected in all the fecal samples, from all animals. Molecular identification using MtDNA (COI) and ribosomal DNA (D2/D3 28S and internal transcribed spacer [ITS] regions) identified the nematode species as Uncinaria hamiltoni. The clinical impacts and the benefits of anthelmintic treatment as a tool for the conservation management of the species are discussed.

New insight into the taxonomic resolution of the genus Bythotrephes Leydig (Crustacea: Cladocera) based on molecular data from Central Europe

The taxonomic status of the genus Bythotrephes Leydig (Crustacea: Cladocera) has been debated since the second half of the XIX century. The most widespread view of recent decades has been that Bythotrephes is a monotypic genus, which was support by preliminary molecular data. However, the recent detailed morphological revision of this genus clearly distinguishes at least seven species. Therefore, we performed a multi-lake survey in Central Europe to give new insight into the taxonomic status of Bythotrephes by combining genetic analysis with traditional morphology-based taxonomy. Based on the morphology we identified two species in Central Europe, B. brevimanus and B. lilljeborgi, as well as hybrid forms. For the genetic analysis, we used newly obtained 113 sequences of mtDNA COI gene of the 535-bp length Bythotrephes from Central Europe and sequences downloaded from GenBank. There were no significant differences between all analyzed sequences, which supports the hypothesis that Bythotrephes is a monotypic genus, with only one highly polymorphic species. On the other hand, the results of our work could point out that the COI gene is insufficient to evaluate the taxonomic status of Bythotrephes. Nonetheless, we have identified 29 new haplotypes of mtDNA COI, and one which was the same as the haplotype found in North America and Finland. Furthermore, this haplotype was the source variant from which most other haplotypes were derived.

Discovery of Root-Lesion Nematode (Pratylenchus scribneri) on Corn in Hainan Province of China

Corn (Zea mays L.) is a very important cereal crop and serves as food, feed, and industrial material (Liu et al. 2016). The root-lesion nematode (RLN) is considered one of the most important plant-parasitic nematodes and can cause economic losses in agriculture worldwide (Jones et al. 2013). In January 2020, five samples were collected from a corn field in Lingshui Lizu Autonomous County, Hainan Province, China. The collected corn plants (cv. Denghai 685) were growing poorly and roots showed distinct lesions and rot. Corn fields with symptoms of stunted plants, and brown lesions on roots were widespread. This corn disease was severe in Lingshui Lizu Autonomous County. RLN were extracted from soil samples by the modified Baermann funnel (Hooper et al. 2005). All the samples contained RLN ranging from 9 to 82 (average 39) RLN per 100 cm3 of soil and 113 to 257 (average 194) RLN per 5 g roots. The extracted RLN were sterilized and cultured on carrot disks at 25°C for 90 days. Afterwards, seeds of corn (cv. Denghai 685) were sown in pots containing 1.8 liters of sterilized soil. Eight plants, one per pot, were infested with 1,000 RLN, eight pots of noninfested corn plants were used as controls, and plants were kept in a greenhouse at 25°C. At 75 days after inoculation, symptoms were like those initially observed in corn fields, whereas no symptoms were observed in the control plants. Nematodes in the soil and roots were extracted using the same method as previously described (Hooper et al. 2005). The average number of RLN per pot was approximately 4,250 in soil and 820 in roots, the reproduction factor (final number of nematodes/initial number of nematodes) was 5.07, no RLN were found in the control. The experiment was conducted twice. The morphological and molecular studies of RLN were examined to confirm species identification. The main morphological measurements of adult females (n = 15) included body length = 526.0 μm ± 17.1 (standard error) (range = 498.0 to 560.5 μm), stylet = 16.0 μm ± 0.3 (15.5 to 16.5 μm), tail length = 29.0 μm ± 1.5 (26.5 to 31.0 μm), a = 23.6 ± 0.6 (22.6 to 24.4), b = 5.6 ± 0.3 (5.2 to 6.0), c = 18.3 ± 0.9 (16.4 to 19.7), V = 78.2% ± 0.6 (77.4 to 79.2%), lip region with two annules. No males were found in the samples. This population was identified as Pratylenchus scribneri, based on the morphological characters (Castillo and Vovlas, 2007). DNA was isolated from individual nematodes followed by proteinase K-based lysis (Wang et al. 2011). The D2/D3 expansion region of the 28S rRNA gene, rDNA-internal transcribed spacer (ITS) region, and mitochondrial cytochrome oxidase I (mtDNA-COI) gene were amplified with primers D2A/-D3B (De Ley et al. 1999), TW81/ AB28 (Vovlas et al. 2011) and JB3/ JB5 (Liu et al. 2018), respectively. The PCR products were purified and ligated into pJET 1.2/blunt cloning vectors and transformed to Escherichia coli strain DH5α, and then sequenced. The obtained 28S rRNA gene D2/D3 region sequences (785bp), ITS sequences (886 bp) and mtDNA-COI (447bp) in this study were submitted to GenBank. The D2/D3 region of the 28S rRNA sequences of the RLN collected in Lingshui (GenBank accession no. MZ701843) showed 99.75% identity with P. scribneri sequences available in the GenBank (KX842628 and KX842625). The ITS sequences of the RLN collected in this study (MZ701842) showed the highest identity of 97.06% with P. scribneri sequences available in the GenBank (KX842626). The mtDNA-COI sequences of the RLN collected in this study (OK040228) showed 100% identity with P. scribneri (MN366409). Both morphological and molecular data confirmed the identity of P. scribneri. P. scribneri has been reported on corn in Inner Mongolia, Hebei, Shanxi, Shandong, Henan, Jiangsu, and Liaoning provinces of China (Li et al. 2019). As far as we know, this is the first report of P. scribneri on corn in Hainan Province, China. Since the RLN can cause considerable damage to corn, strategic measures should be taken to prevent the spread of P. scribneri to other regions in China.