Comparative cytogenetic study of three Macrolophus species (Heteroptera, Miridae.)

Macrolophus pygmaeus (Rambur, 1839) (Insecta, Heteroptera, Miridae) is a predator of key vegetable crop pests applied as a biocontrol agent in the Mediterranean region. M. pygmaeus and M. melanotoma (A. Costa, 1853) are cryptic species with great morphological similarity which results in their misidentification and negative consequences for the conservation of their populations on greenhouse and outdoor crops. In order to find out specific markers for their separation we studied the karyotype, male meiosis and heterochromatin composition of these species and additionally of a third species (as a reference one), M. costalis Fieber, 1858. We demonstrate here that all the three species share achiasmate male meiosis and sex chromosome pre-reduction. On the other hand, the species differ in karyotype, with 2n=28 (26+XY) in M. pygmaeus, 2n=27 (24+X1X2Y) in M. costalis, and 2n=34 (32+XY) in M. melanotoma, and heterochromatin distribution and composition. In addition, the species differ in sperm morphology: sperm cells of M. costalis are significantly longer with longer head and tail than those of M. melanotoma and M. pygmaeus, whereas sperm cells of M. melanotoma have a longer tail than those of M. pygmaeus. All these characters can be used as markers to identify the species, in particular the cryptic species M. melanotoma and M. pygmaeus.This study has been funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (Project AGL2011-24349), and by a travel grant from the University of Lleida - Fundació “La Caixa” to Dr Grozeva. The chromosome analysis was performed using microscope Axio Scope A1 – Carl Zeiss Microscopy upgraded by the project WETLANET (FP7 CSA – SUPPORT ACTION, GA 229802). We thank cordially Prof. Dr V. Kuznetsova for the valuable advices to improve the manuscrip

Elliptic Fourier analysis in the study of the male genitalia to discriminate three Macrolophus species (Hemiptera:Miridae)

Within the genus Macrolophus (Heteroptera: Miridae), the species M. costalis (Fieber), M. melanotoma (Costa) and M. pygmaeus (Rambur) are present in the Mediterranean region on a wide variety of plant species. While M. costalis can easily be separated from the other two by the black tip at the scutellum, M. pygmaeus and M. melanotoma are cryptic species, extremely similar to one another in external traits, which has resulted in misidentifications. M. pygmaeus is an efficient biological control agent, both in greenhouse and field crops. The misidentification of these cryptic species could limit the effectiveness of biological control programs. Although the morphology of the left paramere of the male genitalia has been used as a character for identification of these two cryptic species, there is controversy surrounding the reliability of this character as a taxonomic tool for these species. Using geometric morphometric techniques, which are a powerful approach in detecting slight shape variations, the left parameres from these three Macrolophus species were compared. The paramere of M. costalis was larger and had a different shape to that of M. melanotoma and M. pygmaeus; however, no differences in size or shape were found between the left paramere of M. melanotoma and that of M. pygmaeus. Therefore, our results confirm that this character is too similar and it cannot be used to discriminate between these two cryptic species.This study has been funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (Project AGL2011-24349 and AGL2014-53970-C2-2-R)

Development of specific ITS markers for plant DNA identification within herbivorous insects

DNA based techniques have proved to be very useful methods to study trophic relationships 17 between pests and their natural enemies. However, most predators are best defined as omnivores, 18 and the identification of plant-specific DNA should also allow the identification of the plant 19 species the predators have been feeding on. In this study, a PCR approach based on the 20 development of specific primers was developed as a self-marking technique to detect plant DNA 21 within the gut of one heteropteran omnivorous predator (Macrolophus pygmaeus) and two 22 lepidopteran pest species (Helicoverpa armigera and Tuta absoluta). Specific tomato primers 23 were designed from the ITS 1-2 region, which allowed the amplification of a tomato DNA 24 fragment of 332 bp within the three insect species tested in all cases (100% of detection at t = 0) 25 and did not detect DNA of other plants nor of the starved insects. Plant DNA half-lives at 25ºC 26 ranged from 5.8h, to 27.7h and 28.7h within M. pygmaeus, H. armigera and T. absoluta, 27 respectively. Tomato DNA detection within field collected M. pygmaeus suggests dietary mixing 28 in this omnivorous predator and showed a higher detection of tomato DNA in females and 29 nymphs than males. This study provides a useful tool to detect and to identify plant food sources 30 of arthropods and to evaluate crop colonization from surrounding vegetation in conservation 31 biological control programs

Comparative cytogenetic study of three Macrolophus species (Heteroptera, Miridae.)

Macrolophus pygmaeus (Rambur, 1839) (Insecta, Heteroptera, Miridae) is a predator of key vegetable crop pests applied as a biocontrol agent in the Mediterranean region. M. pygmaeus and M. melanotoma (A. Costa, 1853) are cryptic species with great morphological similarity which results in their misidentification and negative consequences for the conservation of their populations on greenhouse and outdoor crops. In order to find out specific markers for their separation we studied the karyotype, male meiosis and heterochromatin composition of these species and additionally of a third species (as a reference one), M. costalis Fieber, 1858. We demonstrate here that all the three species share achiasmate male meiosis and sex chromosome pre-reduction. On the other hand, the species differ in karyotype, with 2n=28 (26+XY) in M. pygmaeus, 2n=27 (24+X1X2Y) in M. costalis, and 2n=34 (32+XY) in M. melanotoma, and heterochromatin distribution and composition. In addition, the species differ in sperm morphology: sperm cells of M. costalis are significantly longer with longer head and tail than those of M. melanotoma and M. pygmaeus, whereas sperm cells of M. melanotoma have a longer tail than those of M. pygmaeus. All these characters can be used as markers to identify the species, in particular the cryptic species M. melanotoma and M. pygmaeus

Elliptic Fourier analysis in the study of the male genitalia to discriminate three Macrolophus species (Hemiptera:Miridae)

Within the genus Macrolophus (Heteroptera: Miridae), the species M. costalis (Fieber), M. melanotoma (Costa) and M. pygmaeus (Rambur) are present in the Mediterranean region on a wide variety of plant species. While M. costalis can easily be separated from the other two by the black tip at the scutellum, M. pygmaeus and M. melanotoma are cryptic species, extremely similar to one another in external traits, which has resulted in misidentifications. M. pygmaeus is an efficient biological control agent, both in greenhouse and field crops. The misidentification of these cryptic species could limit the effectiveness of biological control programs. Although the morphology of the left paramere of the male genitalia has been used as a character for identification of these two cryptic species, there is controversy surrounding the reliability of this character as a taxonomic tool for these species. Using geometric morphometric techniques, which are a powerful approach in detecting slight shape variations, the left parameres from these three Macrolophus species were compared. The paramere of M. costalis was larger and had a different shape to that of M. melanotoma and M. pygmaeus; however, no differences in size or shape were found between the left paramere of M. melanotoma and that of M. pygmaeus. Therefore, our results confirm that this character is too similar and it cannot be used to discriminate between these two cryptic species