Ma. Teresa Santillán Galicia

Reliable assessment of bee sperm quality is important for breeding programs, artificial insemination, and germplasm conservation. However, incubation conditions for combined fluorescence assessment remain poorly standardized across bee species. This study evaluated a sequential triple-staining protocol using Hoechst 33342, propidium iodide, and FITC-PNA in Apis mellifera and Scaptotrigona mexicana. Five incubation times (5, 10, 15, 20, and 30 min per fluorochrome) were tested. The shortest tested intervals allowing consistent fluorescence interpretation across all five microscopic fields in every biological replicate were 20 min per fluorochrome in A. mellifera and 30 min per fluorochrome in S. mexicana. Using these conditions, sperm vitality and acrosomal integrity were assessed by fluorescence microscopy after one week of storage at 22 °C in FEM7, saline solution (SS), or modified Kiev. Sperm vitality was determined by Hoechst 33342/PI live–dead classification, and localized green FITC-PNA fluorescence was the criterion for acrosomal damage. Binomial generalized linear mixed models were used to analyze the observed counts in relation to species, diluent, and their interaction. A significant species × diluent interaction was detected. Modified Kiev yielded the highest estimated sperm vitality in A. mellifera, without a significant difference from FEM7, whereas FEM7 yielded the highest estimated sperm vitality in S. mexicana. SS yielded the lowest estimated sperm vitality in both species. Acrosomal damage was detected exclusively in spermatozoa classified as dead. These results establish operational staining conditions for the combined assay and show that the relative performance of complete diluent formulations differed between species under the collection and storage conditions tested.

The broad mite Polyphagotarsonemus latus (Banks) is a significant pest of agricultural crops in tropical and subtropical regions (Gerson 1992). It affects economically important plant species such ...

Tetranychus urticae and Tetranychus merganser are economically important phytophagous mites for which sustainable control alternatives are required. Entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, have been proposed as biological control agents. However, information on stage-specific susceptibility and reproductive effects in these species remains limited. This study evaluated the susceptibility of eggs, larvae, nymphs and adult females of T. urticae and T. merganser to isolates of M. anisopliae and B. bassiana under laboratory conditions, and assessed their effects on oviposition and offspring sex ratio. Eggs and larvae of both mite species showed low susceptibility, with no significant differences between mite species or fungal isolate; mortality never exceeding 20%, indicating limited efficacy against early developmental stages of mites. At the nymphal stage, significant differences were detected only in T. urticae, where isolate Ma129 (M. anisopliae) caused higher mortality than B. bassiana isolate Bb88, although overall mortality remained below 20%. In contrast, adult females were the most susceptible stage, exhibiting approximately 70% mortality, with no significant differences between mite species or fungal isolates. Fecundity differed significantly between mite species; T. urticae females produced more eggs than T. merganser. However, within each mite species, fungal isolates did not significantly affect oviposition compared with the control. Female longevity was the principal determinant of total fecundity, with an estimated increase of 6.98 ± 0.17 eggs per additional day of survival, confirming the strong relationship between lifespan and reproductive output. Offspring sex ratio was female-biased (> 70%) and not influenced by fungal isolate or mite species, although it varied significantly over time. These results demonstrate clear stage-dependent susceptibility, with adult females representing the most vulnerable stage, while early developmental stages exhibited substantial tolerance. Under the conditions tested, the fungal isolates did not induce detectable sublethal effects on reproductive output or progeny sex ratio, highlighting the importance of targeting life-stage when incorporating entomopathogenic fungi into mite management strategies.

To evaluate the effect of mite density of B. yothersi and B. californicus on acquisition and transmission of Cilevirus leprosis. To determine the effect of mite density on virus acquisition, three densities of B. yothersi (5, 10, and 15 adult females) were exposed to a virus source (orange fruits exhibiting symptoms of Cilevirus leprosis) for seven days. For B. californicus only the 15-mite density was evaluated. Control treatments consisted of the same densities for both mite species; however, in these treatments, mites were allowed to feed on non-infected oranges. For transmission experiment, only B. yothersi was used. Mites were exposed to a virus source as described previously, using the same densities (5, 10, and 15 adult females), each with its corresponding control treatment. After seven days of feeding, all mites were transferred to seven-day-old bean plants (Phaseoulus vulgaris var. Jamapa). The proportion of mites positive for the virus in each treatment was determined using RT-PCR. RNA from each individual mite was extracted using the ZR Tissue andamp; Insect RNA MicroPrep kit, and cDNA was synthesised using Sensiscript. Conventional PCR assays to detect the virus were done using the primers of Locali et al. (2003). For virus quantification in mites from each treatment, a TaqMan probe and one set of primers were designed using the movement protein gene region of the complete and partial RNA2 sequences of CiLV-C. The primers (F: TGA CAC CGA ACG ATA TGA TTT CTC, R: ATA TCT GCC TAA AAG ACT CCA CAG) and the TaqMan probe (CCC CAG CGT CTT CAG CTT TAC GGT) were designed on the software AlleleID 6 and synthesized. FAM and BHQ1 were used to label the probe at the 5' and 3' end, respectively. For the bean leaves, RNA was extracted from 10 mg of leaf tissue using the Quick-RNA Plant Miniprep kit, and virus quantities were estimated as describes previously. Mite density did not affect the proportion of B. yothersi mites acquiring Cilevirus leprosis but affected the quantity of virus acquired. We obtained 100% infection of bean leaves, although no significant differences in viral quantity were observed among treatments. B. californicus did not acquire the virus. Our findings provided new insights into the effect of mite-virus interactions in the transmission of this virus.

The phytophagy, biology, and thermal requirements of Tarsonemus confusus Ewing on strawberry were determined, together with the damage caused to flowers and fruits at different population densities under controlled conditions. In addition, a deep learning tool for early mite diagnosis was developed, based on the detection of tarsonemid-associated damage using the YOLOv11 model developed by Ultralytics, and validated in commercial fields. Mites were collected from a commercial strawberry crop in Michoacán, Mexico (20°06'75.11"N, -102°38'78.35"W). A mite colony of T. confususwas maintained on strawberry flowers under laboratory conditions (25°C, 65% RH, and a 12:12 h L:D). Identification was performed using morphological characters based on the taxonomic keys of Lindquist (1986) and Nucifora andamp; Vacante (2004). Biology, thermal requirements, and reproductive behaviour were determined at three temperature regimes (20, 25, and 28 °C) under a completely randomized design. Developmental times, lower developmental thresholds, and thermal constants were estimated using mathematical models and linear regression analyses. To assess damage, mite-free strawberry plants were inoculated with different population densities. Injuries to flowers and fruits were recorded, including blight- like symptoms on stamens and carpels, brown spotting on petals, deformities, and achene disorganization. Population density was quantified, and data were statistically analysed. Field sampling and image acquisition were also conducted using a Phantom 4 Pro V2.0 drone to validate mite presence in commercial fields. Finally, a deep-learning tool was developed using the YOLOv11Ultralytics model, based on both field and laboratory images for early damage detection. The dataset was integrated, labeled, and scaled to train a system capable of recognizing symptoms Tarsonemus confusus was found associated with strawberry flowers and fruits. Its development was significantly influenced by temperature, with the duration of the egg, larvae, nymph, and adult stages decreasing as temperature increased from 20 to 28 °C. Base temperatures ranged from 9.2 to 14.6 °C and stage-specific thermal constants were estimated, showing a linear relationship between temperature and developmental rate. Artificial infestations caused severe damage to flowers and fruits, including necrosis, deformities, and quality loss. Based on these symptoms, an image dataset was generated to train a deep learning model based on YOLOv11, with a recall of 0.85, mAP of 0.74, and precision of 0.73 aimed at early diagnosis under field conditions. Our study contributes new insights into the feeding behaviour habits of T. confusus and contributes to the development of novel technologies for early pest diagnosis.

Objective: To evaluate the water expenditure of two strawberry cultivars (Albion and San Andreas) inoculated with three arbuscular mycorrhizal fungi (AMF) grown in two substrates. Moreover, to determine their efficiency in the strawberry plant nutrition and fruit quality. Design/ Methodology/ Approach: Five treatments were established: plants inoculated with Funneliformis geosporum, F. mosseae BEG25, and F. mosseae Mich; with conventional management (M. conv), and control. Water consumption, photosynthetic pigments, plant macro- and micronutrient concentrations, soluble sugar concentration, titratable acidity, pH, and Brix degrees in fruits, and mycorrhizal colonization and glomalin concentration were analyzed. Results: In both substrates, the lowest water consumption was observed in plants inoculated with F. mosseae Mich. Plants inoculated with F. mosseae BEG25 had similar foliar and fruit concentrations of almost all nutrients analyzed to those of plants with the M. conv treatment. Fruits from inoculated plants had higher Brix degrees, total sugars, glucose, and citric acid than these parameters in control plants. Limitations/ Implications of the study: Although inoculated plants received 40% less nutrient solution (with fertilizer cost savings of US$461 per ha), these showed improved plant nutrition and fruit quality. Findings/ Conclusions: AMF inoculation is a promising strategy for strawberry production under greenhouse conditions, with savings in water and fertilizer use.

This study compared the life-history traits and host-plant interactions of Tetranychus merganser and T. urticae. It provides a comprehensive assessment of both species by integrating taxonomic, demographic, and host plant interaction data. Morphological identification based on the shape of the aedeagus was supported by molecular analysis of partial COI sequences. Morphometric analyses revealed that all aedeagal structures and several somatic measurements were significantly larger in T. merganser males, whereas T. urticae females had longer and wider idiosomal dimensions. Demographically, T. merganser exhibited a significantly longer developmental time across most immature stages and higher adult survivorship, indicating slower development compared with T. urticae, while T. merganser showed greater mean generation and doubling times, reflecting slower population growth but longer longevity. The total number of eggs laid per female and sex ratios did not differ significantly between species, although both were strongly female-biased. Host-plant experiments demonstrated species-specific preferences, T. urticae attained higher populations and caused greater foliar damage on rose, while T. merganser performed better on pepper. Overall, these results highlight clear ecological differentiation between T. merganser and T. urticae, supporting their status as an important pest species with differing life-history and host-plant adaptation strategies.

Citrus leprosis is a non-systemic disease caused by citrus leprosis virus (CiLV), which is classified as cytoplasmic (CiLV-C) or nuclear (CiLV-N) based on its replication site within host cells. Mite species in the genus Brevipalpus vector this virus. In Mexico, B. californicus and B. yothersi have been recorded in citrus orchards, with the latter species being most abundant and widely distributed. Despite extensive research, knowledge gaps remain regarding interactions between Brevipalpus and CiLV-C, the predominant virus type. We investigated the vector competence of both species, with a detailed analysis of density-dependent acquisition and transmission for B. yothersi . Virus acquisition was assessed using three densities (5, 10, or 15 adult females) for B. yothersi and a single desity (15 mites) for B. californicus . Virus detection and quantification were performed using a TaqMan probes targeting the viral movement protein gene. Transmission assays were conducted on Phaseolus vulgaris plants using viruliferous B. yothersi at all densities. B. californicus did not acquire the virus and was therefore excluded from transmission experiments. In contrast, B. yothersi successfully acquired the virus at all densities. While the proportion of viruliferous mites did not differ significantly among density treatments, viral load per mite was significantly higher at the lowest density. Virus transmission ocurred at all densities, with no significant differences in viral titres in inoculated plants. There results provide insights into density-related mite-virus interactions affecting CiLV-C transmission.

Citrus leprosis virus C (CiLV-C) (Cilevirus) and orchid fleck virus citrus strain (OFV-Cit) (Dichorhavirus) are viruses associated with citrus leprosis disease. Although symptoms associated with CiLV-C were observed in orange in 2005 in Mexico, and confirmed using molecular techniques in 2011, no studies have been made on the distribution of either CiLV-C or OFV-Cit viruses. During 2017, we studied the geographical distribution and host range of these two viruses infecting citrus orchards in Mexico, specifically orange, lime, mandarin and grapefruit orchards in 15 Mexican states. Furthermore, in 2019 we sampled lime orchards in three Mexican states. Presence of CiLV-C and OFV-Cit was determined using reverse transcription polymerase chain reaction (RT-PCR) assays. During 2017 the proportion of leaves infected by either CiLV-C or OFV-Cit was significantly affected by geographical origin. However, only a few samples were obtained from mandarin and grapefruit so these data were excluded from statistical analysis; orange had significantly higher rates of infection with CiLV-C than lime and the opposite was observed for OFV-Cit. Using RT-PCR, some asymptomatic leaves from 2017 samples were positive for the viruses of interest. In 2019 no symptoms associated with leprosis were observed in any of the leaves sampled from lime orchards. However, low infection rates were detected, with 6% of samples testing positive for CiLV-C and 3% for OFV-Cit. To confirm the identity of the CiLV-C isolate found in lime leaves collected in 2019, we sequenced nearly the complete RNA2 genomic region of the virus. Basic Local Alignment Search Tool (BLAST) search revealed 98.99% similarity with previously reported CiLV-C sequences from other citrus species. The implications of our results for field monitoring and disease detection are discussed.

BACKGROUND: Most studies on efficacy of fungal pathogens and predatory mites against Tetranychus urticae have been done on individual species in the laboratory. We evaluated fungi and predatory mites separately and together against glasshouse populations of T. urticae on chrysanthemum plants. First, effectiveness of the fungal pathogens Beauveria bassiana (Bb88) and Metarhizium anisopliae (Ma129) was compared; then, effectiveness of the predatory mites Phytoseiulus persimilis and Neoseiulus californicus. Based on the results, N. californicus and isolate Ma129 were selected and evaluated in combination. In all experiments, treatment effects were assessed for eggs and motile stages of T. urticae.RESULTS: The first experiment showed no significant effect of either fungal isolate on T. urticae populations, except on plants initially infested with 20 mites, where more eggs were found in the control compared to the fungal treatments. In the second experiment, both predatory mites were equally effective at reducing T. urticae populations compared with the control, regardless of initial T. urticae population density. The last experiment demonstrated that populations of T. urticae were reduced most when M. anisopliae (Ma129) and N. californicus were applied together, compared with the control and when each natural enemy was applied separately.CONCLUSIONS: Metarhizium anisopliae (Ma129) and B. bassiana (Bb88) isolates did not have a significant effect on reducing T. urticae populations. Both predatory mites reduced T. urticae populations, regardless of T. urticae density. Combined application of M. anisopliae (Ma129) and N. californicus were more effective against T. urticae than the control or when each agent was applied separately. © 2024 Society of Chemical Industry.