Behaviour, feeding and cytogenetic features of the wingless blood-sucking ectoparasite Cyanolicimex patagonicus (Heteroptera: Cimicidae).

Cyanolicimex (Haematosiphoninae) includes a single species, C. patagonicus, which is found in the largest known colony of its avian host Cyanoliseus patagonus (Psittacidae) located in Patagonia (Argentina). Relationships between Cyanolicimex and other genera of Haematosiphoninae are still unclear because this genus shares some characters with other South American genera and possesses some similarities with Hesperocimex from the Neoarctic region. The aim of the present study was to provide additional data of C. patagonicus so as to better understand its relationships with other South American species. We examined some biological features of C. patagonicus in the field and we performed a cytogenetic analysis. We observed in the field that C. patagonicus does not live inside the hollow nests of Cyanoliseus patagonus. The cytogenetic analysis showed that the male karyotype is 2n= 31= 28A+X1X2Y and revealed an achiasmate male meiosis and of the collochore type. Our results together with available cytogenetic data in other cimicids, allow proposing the possible chromosomal rearrangements involved in the chromosomal evolution of C. patagonicus and also contribute to better understand the evolutionary divergence at the chromosomal level within Haematosiphoninae. Based on the whole evidence, we propose to place in four groups the species of Haematosiphoninae cytogenetically hitherto studied.

Unusual chromosome numbers and polyploidy in invasive fire ant populations.

Fire ants (Solenopsis invicta Buren in J Ga Entomol Soc 7:1-26, 1972), an invasive alien ant species, first spread from South America to the United States in the 1930s, the southern part of the United States by the end of the twentieth century, Oceania, Taiwan, and China in the twenty-first century, and finally to Japan and South Korea in 2017. As these ants have significant negative economic, human health, and environmental impacts, the purpose of this research was to accumulate cytogenetic information regarding fire ants and provide basic data for developing management strategies for their control. Fire ants were collected from invasive populations from Taiwan, Florida (USA), and Buenos Aires (Argentina), and a native population from Puerto Iguazu (Argentina), their point of origination, and analyzed with regard to chromosome number, morphology, and polyploidy, silver-stained nucleolar organizer regions (Ag-NORs), and 18S rDNA and telomere fluorescence in situ hybridization (FISH). The results showed that (1) fire ants from invaded populations differed in chromosome morphology compared to those from native populations; (2) the Florida and Taiwanese fire ant populations evinced greater variability in chromosome numbers and polyploidy variations; (3) the Taiwanese population exhibited significantly increased Ag-NOR signals in interphase cells, with signal number significantly positively correlating with distance from native populations; and (4) substantial diversity of signals was also apparent following 18S rDNA and telomere FISH analyses. Variation in these characteristics were hypothesized to be due to (1) the effect of hybridizations and interbreeding between closely related species or genetically distant populations, and (2) the potential effect of large amounts of insecticides sprayed for pest control.

A karyotype comparison between two species of bordered plant bugs (Hemiptera, Heteroptera, Largidae) by conventional chromosome staining, C-banding and rDNA-FISH.

A cytogenetic characterization, including heterochromatin content, and the analysis of the location of rDNA genes, was performed in Largus fasciatus Blanchard, 1843 and L. rufipennis Laporte, 1832. Mitotic and meiotic analyses revealed the same diploid chromosome number 2n = 12 + X0/XX (male/female). Heterochromatin content, very scarce in both species, revealed C-blocks at both ends of autosomes and X chromosome. The most remarkable cytological feature observed between both species was the different chromosome position of the NORs. This analysis allowed us to use the NORs as a cytological marker because two clusters of rDNA genes are located at one end of one pair of autosomes in L. fasciatus, whereas a single rDNA cluster is located at one terminal region of the X chromosome in L. rufipennis. Taking into account our results and previous data obtained in other heteropteran species, the conventional staining, chromosome bandings, and rDNA-FISH provide important chromosome markers for cytotaxonomy, karyotype evolution, and chromosome structure and organization studies.

Chromosomal distribution of interstitial telomeric sequences as signs of evolution through chromosome fusion in six species of the giant water bugs (Hemiptera, Belostoma).

Tandem arrays of TTAGG repeats show a highly conserved location at the telomeres across the phylogenetic tree of arthropods. In giant water bugs Belostoma, the chromosome number changed during speciation by fragmentation of the single ancestral X chromosome, resulting in a multiple sex chromosome system. Several autosome-autosome fusions and a fusion between the sex chromosome pair and an autosome pair resulted in the reduced number in several species. We mapped the distribution of telomeric sequences and interstitial telomeric sequences (ITSs) in Belostoma candidulum (2n = 12 + XY/XX; male/female), B. dentatum (2n = 26 + X1X2Y/X1X1X2X2), B. elegans (2n = 26 + X1X2Y/X1X1X2X2), B. elongatum (2n = 26 + X1X2Y/X1X1X2X2), B. micantulum (2n = 14 + XY/XX), and B. oxyurum (2n = 6 + XY/XX) by FISH with the (TTAGG) n probes. Hybridization signals confirmed the presence of TTAGG repeats in the telomeres of all species examined. The three species with reduced chromosome numbers showed additional hybridization signals in interstitial positions, indicating the occurrence of ITS. From the comparison of all species here analyzed, we observed inverse relationships between chromosome number and chromosome size, and between presence/absence of ITS and chromosome number. The ITS distribution between these closely related species supports the hypothesis that several telomere-telomere fusions of the chromosomes from an ancestral diploid chromosome number 2n = 26 + XY/XX played a major role in the karyotype evolution of Belostoma. Consequently, our study provide valuable features that can be used to understand the karyotype evolution, may contribute to a better understanding of taxonomic relationships, and also elucidate the high plasticity of nuclear genomes at the chromosomal level during the speciation processes.

Genotoxic effects of Roundup Full II® on lymphocytes of Chaetophractus villosus (Xenarthra, Mammalia): In vitro studies.

In Argentina, Chaetophractus villosus has a wide distribution that overlaps with agricultural areas where soybean is the predominant crop. In such areas the pesticide Roundup Full II® (RU) is widely applied. The genotoxic effect of its active ingredient glyphosate (RU is 66.2% glyphosate) on the peripheral blood lymphocytes of C. villosus was tested over a range of concentrations (280, 420, 560, 1120 µmol/L). Culture medium without glyphosate served as negative control, while medium containing mitomycin C served as positive control. Genetic damage was characterized in terms of the percentage of cells with chromosome aberrations (CA), the mean number of sister chromatid exchanges (SCE) per cell, and the modification of cell proliferation kinetics via the calculation of the replication index. Significant increases (p < 0.0001) were seen in the CA frequency and the mean number of SCEs per cell compared to negative controls at all the RU concentrations tested. Chromatid breaks, the only form of CA observed, under the 560 µmol/L RU conditions and in presence of mitomycin C were four to five times more common than at lower concentrations, while no viable cells were seen in the 1120 µmol/L treatment. The mean number of SCEs per cell was significantly higher under the 280 µmol/L RU conditions than the 420 or 560 µmol/L RU conditions; cells cultivated in the presence of MMC also showed significantly more SCEs. All the RU concentrations tested (except in the 1120 µmol/L RU treatment [no viable cells]) induced a significant reduction in the replication index (p < 0.0001). The present results confirm the genotoxic effects of RU on C. villosus lymphocytes in vitro, strongly suggesting that exposure to RU could induce DNA damage in C. villosus wildlife.

Chaetophractus villosus as a sentinel organism: Baseline values of mitotic index, chromosome aberrations and sister chromatid exchanges.

Sentinel species are useful tools for studying the deleterious effects of xenobiotics on wildlife. The large hairy armadillo (Chaetophractus villosus) is the most abundant and widely distributed mammal in Argentina. It is a long-lived, omnivorous, burrowing species, with fairly restricted home ranges. To evaluate the level of spontaneous genetic damage in this mammal, we determined the baseline values of several genotoxicity biomarkers. The study included 20 C. villosus adults of both sexes from eight pristine localities within its geographic distribution range. Genotoxicity analysis was performed on 72-h lymphocyte cultures, using mitomycin C as positive control. We obtained the baseline values of mitotic index (MI=10.52±0.30 metaphases/total cells, n=20), chromosome aberrations (CA=0.13±0.22, n=20), sister chromatid exchanges (SCE)=6.55±0.26, n=6) and replication index (RI=1.66, n=6). MI and CA did not show significant differences (P>0.05) among localities or between sexes. No significant differences in MI, CA, SCE, and RI (P>0.05) were found between values from the pristine localities and historical data. There were significant differences in CA, SCE, and RI (P<0.05) between lymphocyte cultures from pristine localities and those exposed to mitomycin C. We propose the large hairy armadillo as a sentinel organism for environmental biomonitoring of genotoxic chemicals due to its abundance, easy manipulation, well-known biology, the fact that it is usually exposed to different mixtures and concentrations of environmental contaminants, and the baseline values of genetic damage characterized by MI, CA, SCE and RI as biomarkers.

Comparative study of mitotic chromosomes in two blowflies, Luciliasericata and L.cluvia (Diptera, Calliphoridae), by C- and G-like banding patterns and rRNA loci, and implications for karyotype evolution.

The karyotypes of Luciliacluvia (Walker, 1849) and Luciliasericata (Meigen, 1826) from Argentina were characterized using conventional staining and the C- and G-like banding techniques. Besides, nucleolus organizer regions (NORs) were detected by fluorescent in situ hybridization (FISH) and silver staining technique. The chromosome complement of these species comprises five pairs of autosomes and a pair of sex chromosomes (XX/XY, female/male). The autosomes of both species have the same size and morphology, as well as C- and G-like banding patterns. The X and Y chromosomes of Luciliacluvia are subtelocentric and easily identified due to their very small size. In Luciliasericata, the X chromosome is metacentric and the largest of the complement, showing a secondary constriction in its short arm, whereas the Y is submetacentric and smaller than the X. The C-banding patterns reflect differences in chromatin structure and composition between the subtelocentric X and Y chromosomes of Luciliacluvia and the biarmed sex chromosomes of Luciliasericata. These differences in the sex chromosomes may be due to distinct amounts of constitutive heterochromatin. In Luciliacluvia, the NORs are placed at one end of the long-X and of the long-Y chromosome arms, whereas one of the NORs is disposed in the secondary constriction of the short-X chromosome arm and the other on the long-Y chromosome arm in Luciliasericata. Although the G-like banding technique does not yield G-bands like those in mammalian chromosomes, it shows a high degree chromosomal homology in both species because each pair of autosomes was correctly paired. This chromosome similarity suggests the absence of autosomal rearrangements during karyotype evolution in the two species studied.

The significance of cytogenetics for the study of karyotype evolution and taxonomy of water bugs (Heteroptera, Belostomatidae) native to Argentina.

Male meiosis behaviour and heterochromatin characterization of three big water bug species were studied. Belostoma dentatum (Mayr, 1863), Belostoma elongatum Montandon, 1908 and Belostoma gestroi Montandon, 1903 possess 2n = 26 + X1X2Y (male). In these species, male meiosis is similar to that previously observed in Belostoma Latreille, 1807. In general, autosomal bivalents show a single chiasma terminally located and divide reductionally at anaphase I. On the other hand, sex chromosomes are achiasmatic, behave as univalents and segregate their chromatids equationally at anaphase I. The analysis of heterochromatin distribution and composition revealed a C-positive block at the terminal region of all autosomes in Belostoma dentatum, a C-positive block at the terminal region and C-positive interstitial dots on all autosomes in Belostoma elongatum, and a little C-positive band at the terminal region of autosomes in Belostoma gestroi. A C-positive band on one bivalent was DAPI negative/CMA3 positive in the three species. The CMA3-bright band, enriched in GC base pairs, was coincident with a NOR detected by FISH. The results obtained support the hypothesis that all species of Belostoma with multiple sex chromosome systems preserve NORs in autosomal bivalents. The karyotype analyses allow the cytogenetic characterization and identification of these species belonging to a difficult taxonomic group. Besides, the cytogenetic characterization will be useful in discussions about evolutionary trends of the genome organization and karyotype evolution in this genus.

Pseudacteon tricuspis: its behavior and development according to the social form of its host and the role of interference competition among females.

We studied how the behavior and performance of Pseudacteon tricuspis Borgmeier varies with the social form of its host Solenopsis invicta Buren, in its native range in Argentina where monogyne colonies are more abundant than polygynes (approximately 75 vs. 25%). Female, P. tricuspis took 44% less time (50 vs. 89 s) to attack monogyne than polygyne ants, but oviposition attempts were similar (23 vs. 18 attacks). The presence of the parasitoid affected the average size of foragers on the trail, with the proportion of minor workers increasing on both social forms. In the laboratory, P. tricuspis selected similar host sizes, although pupal survival was 25% higher on monogynes than on polygynes. Developmental times of both genders were similar (33-35 d), although larger females emerged from bigger hosts. The sex ratio of P. tricuspis was more male biased when exploiting polygyne ants. Intraspecific competition significantly affected parasitoid reproductive success, being significantly higher for a solitary female than when three females were present, although the size of workers selected did not vary. The male:female ratio also changed, being 1:1 without competition but 2:1 with competition. We demonstrated for the first time the consequences of interference competition among P. tricuspis females, a common behavior observed in others parasitoids. We discuss why P. tricuspis sex ratios are always biased toward males in both social forms and suggest that similar studies of interference competition within and between already naturalized Pseudacteon species in the United States could help predict establishment patterns.

Behavior and development of Pseudacteon curvatus (Diptera: Phoridae) varies according to the social form of its host Solenopsis invicta (Hymenoptera: Formicidae) in its native range.

We assessed the performance of Pseudacteon curvatus Borgmeier with respect to the social form of Solenopsis invicta Buren in Argentina In the field, we studied the effect the parasitoid on size and proportion of ant foragers. In the laboratory, we evaluated P. curvatus oviposition preferences; host size elected; developmental periods; and sexual size dimorphism, sex ratio, and parasitoid survivorship. P. curvatus affected the average size of foraging workers on both social forms diminishing the proportion of big and increasing the proportion of minor workers. P. curvatus required a shorter orientation time and exhibited a greater number of attacks when ovipositing on monogynes workers. In the laboratory, host sizes elected by P. curvatus were similar between social forms. However, attacks on polygyne colonies were more variable, increasing the number of unviable offspring. Developmental times of females and males of P. curvatus were similar for both social forms, but total developmental periods were shorter for males from monogyne colonies. We did not find differences between sexes in emerging adults' size by social form and the female: male sex ratio was 1:1 for both social forms. P. curvatus pupae survival and adult emergence per trial from monogyne colonies were greater than from polygyne colonies. The rarity of polygyne S. invicta in its native range may prevent this phorid from adjusting its life history to that social form. Consequences of applying this phorid in biological control are discussed.

Cytogenetic analysis of three species of Pseudacteon (Diptera, Phoridae) parasitoids of the fire ants using standard and molecular techniques.

Pseudacteon flies, parasitoids of worker ants, are being intensively studied as potentially effective agents in the biological control of the invasive pest fire ant genus Solenopsis (Hymenoptera: Formicidae). This is the first attempt to describe the karyotype of P. curvatus Borgmeier, P. nocens Borgmeier and P. tricuspis Borgmeier. The three species possess 2n = 6; chromosomes I and II were metacentric in the three species, but chromosome pair III was subtelocentric in P. curvatus and P. tricuspis, and telocentric in P. nocens. All three species possess a C positive band in chromosome II, lack C positive heterochromatin on chromosome I, and are mostly differentiated with respect to chromosome III. P. curvatus and P. tricuspis possess a C positive band, but at different locations, whereas this band is absent in P. nocens. Heterochromatic bands are neither AT nor GC rich as revealed by fluorescent banding. In situ hybridization with an 18S rDNA probe revealed a signal on chromosome II in a similar location to the C positive band in the three species. The apparent lack of morphologically distinct sex chromosomes is consistent with proposals of environmental sex determination in the genus. Small differences detected in chromosome length and morphology suggests that chromosomes have been highly conserved during the evolutionary radiation of Pseudacteon. Possible mechanisms of karyotype evolution in the three species are suggested.

Cytogenetic analysis of three species of Pseudacteon (Diptera, Phoridae) parasitoids of the fire ants using standard and molecular techniques

Pseudacteon flies, parasitoids of worker ants, are being intensively studied as potentially effective agents in the biological control of the invasive pest fire ant genus Solenopsis (Hymenoptera: Formicidae). This is the first attempt to describe the karyotype of P. curvatus Borgmeier, P. nocens Borgmeier and P. tricuspis Borgmeier. The three species possess 2n = 6; chromosomes I and II were metacentric in the three species, but chromosome pair III was subtelocentric in P. curvatus and P. tricuspis, and telocentric in P. nocens. All three species possess a C positive band in chromosome II, lack C positive heterochromatin on chromosome I, and are mostly differentiated with respect to chromosome III. P. curvatus and P. tricuspis possess a C positive band, but at different locations, whereas this band is absent in P. nocens. Heterochromatic bands are neither AT nor GC rich as revealed by fluorescent banding. In situ hybridization with an 18S rDNA probe revealed a signal on chromosome II in a similar location to the C positive band in the three species. The apparent lack of morphologically distinct sex chromosomes is consistent with proposals of environmental sex determination in the genus. Small differences detected in chromosome length and morphology suggests that chromosomes have been highly conserved during the evolutionary radiation of Pseudacteon. Possible mechanisms of karyotype evolution in the three species are suggested.